Ribonuclease resistant RNA preparation and utilization

ABSTRACT

The present invention relates to nuclease resistant nucleic acids in general and ribonuclease resistant RNAs in particular. Methods of making and using such nucleic acids are disclosed.

BACKGROUND OF THE INVENTION

This is a continuation-in-part of co-pending application Ser. No.08/675,153, filed Jul. 3, 1996, and a continuation-in-part of co-pendingprovisional application Ser. No. 60/021,145, filed Jul. 3, 1996.

In the last few years, diagnostic assays and assays for specific mRNAspecies have been developed based on the detection of specific nucleicacid sequences. These assays depend on such technologies as RT-PCR™(Mulder, 1994), isothermal amplification (NASBA) (Van Gemen, 1994), andbranched chain DNA (Pachl, 1995). Many of these assays have been adaptedto determine the absolute concentration of a specific RNA species. Theseabsolute quantification assays require the use of an RNA standard ofwhich the precise amount has been previously determined. These RNAstandards are usually synthesized by in vitro transcription or are theinfectious agents themselves. The RNA is purified and then quantified byseveral different methods, such as absorbance at OD₂₆₀, phosphateanalysis, hyperchromicity or isotopic tracer analysis (Collins, 1995).

Quantifying virus RNA sequences in plasma is an important tool forassessing the viral load in patients with, for example, HumanImmunodeficiency Virus (HIV), Hepatitis C Virus (HCV), and other virusessuch as HTLV-1, HTLV-2, hepatitis G, enterovirus, dengue fever virus,and rabies. Viral load is a measure of the total quantity of viralparticles within a given patient at one point in time. In chronicinfections viral load is a function of a highly dynamic equilibrium ofviral replication and immune-mediated host clearance. The benefits ofdetermining viral load include the ability to: 1) assess the degree ofviral replication at the time of diagnosis—an estimate having prognosticimplications, 2) monitor the effect of antiviral medications early inthe disease course, and 3) quickly assess the effects of changingantiviral medications.

Presently, the most sensitive method available for HIV quantification inplasma employs PCR™. There are 4 major steps involved in PCR™ analysisof HIV: 1) Sample preparation, 2) Reverse transcription, 3)Amplification, and 4) Detection. Variability in any of these steps willaffect the final result. An accurate quantitative assay requires thateach step is strongly controlled for variation. In the more rigorousPCR™ assay formats, a naked RNA standard is added to the denaturant justprior to the isolation of the viral RNA from plasma (Mulder, 1994). Aless precise method is to add the standard to the viral RNA after it hasbeen purified (Piatak, 1993). It is important that the RNA s standardsare precisely calibrated and that they withstand the rigors of the assayprocedures.

There is a need for ribonuclease resistant RNA standards. RNA issusceptible to environmental ribonucleases. Producing ribonuclease-freereagents is non-trivial. A danger in using naked RNA as a standard forquantification is its susceptibility to ribonuclease digestion.Compromised standards generate inaccurate values. This problem can becompounded in clinical laboratory settings where the personnel are notusually trained in RNA handling. These factors introduce doubt as to thevalidity of the data generated.

Naked RNA standards are very susceptible to ribonuclease digestion. SomeRNA based assays have been formatted so that users access an RNAstandard tube only once and then discard it to minimize the possibilityof contaminating the RNA standard with ribonucleases. However, thestandards are aliquoted into microfuge tubes which are not guaranteed tobe ribonuclease-free introducing another potential source forcontamination. As well, there is a short period of time during which theRNA is exposed to a pipette tip before it is placed in the denaturingsolution. If the pipette tip is contaminated with ribonuclease then theRNA standard will be degraded and the assay compromised. Anotherdisadvantage of using naked RNA standards are that they must be storedfrozen. In the branched DNA HIV assay formatted by Chiron Corp., thepotential for RNA degradation is so risky that their assays includesingle stranded DNA instead of RNA for their standard (Pachl, 1995). TheDNA is calibrated against RNA. The DNA standard is much less likely tobe degraded. Thus, there is a need for RNA standards which are resistantto ribonucleases and in which there is no doubt about the integrity ofthe standard. These standards would also be more convenient if they didnot need to be stored frozen so that they could be used immediately, nothawing required.

Those of skill know how to bring about chemical alteration of RNA. Suchalterations can be made to nucleotides prior to their incorporation intoRNA or to RNA after it has been formed. Ribose modification (Piecken1991) and phosphate modification (Black, 1972) have been shown toenhance RNA stability in the presence of nucleases. Modifications of the2′ hydroxyl and internucleotide phosphate confers nuclease resistance byaltering chemical groups that are necessary for the degradationmechanism employed by ribonucleases (Heidenreich, 1993). While suchchemical modification can confer ribonuclease resistance, there is noknown suggestion in the art that such ribonuclease resistant structurescould be useful as RNA standards.

RNA bacteriophages have long been used as model systems to study themechanisms of RNA replication and translation. The RNA genome within RNAbacteriophages is resistant to ribonuclease digestion due to the proteincoat of the bacteriophage. Bacteriophage are simple to grow and purify,and the genomic RNA is easy to purify from the bacteriophages. Thesebacteriophages are classified into subgroups based on serotyping.Serologically, there are four subclasses of bacteriophage, whilegenetically, there are two major subclasses, A and B (Stockley, 1994;Witherell, 1991). Bacteriophage MS2/R17 (serological group I) have beenstudied extensively. Other well-studied RNA bacteriophages include GA(group II), Q-beta (group III), and SP (group IV). The RNAbacteriophages only infect the male strains of Escherichia coli, thatis, those which harbor the F′ plasmid and produce an F pilus forconjugation.

The MS2 bacteriophage is an icosahedral structure, 275 Å in diameter,and lacks a tail or any other obvious surface appendage (Stockley,1994). The bacteriophage has large holes at both the 5- and 3-fold axeswhich might be the exit points of the RNA during bacterial infection.The MS2 bacteriophage consists of 180 units of the bacteriophage CoatProtein (˜14 kDa) which encapsidate the bacteriophage genome (seereviews, Stockley, 1994; Witherell, 1991). The MS2 RNA genome is asingle strand encoding the (+) sense of 3569 nucleotides. The genes areorganized from the 5′ end as follows: the Maturase or A protein, thebacteriophage Coat Protein, a 75 amino acid Lysis Protein, and aReplicase subunit. The Lysis gene overlaps the Coat Protein gene and theReplicase gene and is translated in the +1 reading frame of the CoatProtein. Each bacteriophage particle has a single copy of Maturase whichis required for interacting with the F pilus and thus mediatingbacterial infection.

Packaging of the RNA genome by Coat Protein is initiated by the bindingof a dimer of Coat Protein to a specific stem-loop region (the Operatoror “pac” site) of the RNA genome located 5′ to the bacteriophageReplicase gene. This binding event appears to trigger the completeencapsidation process. The sequence of the Operator is not as criticalas the stem-loop structure. The Operator consists of 21 nucleotides andonly two of these residues must be absolutely conserved for Coat Proteinbinding.

The viral Maturase protein interacts with the bacteriophage genomic RNAat a minimum of two sites in the genome (Shiba, 1981). It is evidentlynot required for packaging. However, its presence in the bacteriophageparticle is required to preserve the integrity of the genomic RNAagainst ribonuclease digestion (Argetsinger, 1966; Heisenberg, 1966).

Attempts to produce a viable, infectious recombinant RNA (reRNA)bacteriophage have been unsuccessful. The bacteriophage are veryefficient at deleting heterologous sequences and the fidelity of theReplicase is poor such that point mutations occur at the rate of˜1×10⁻⁴.

Pickett and Peabody (1993) performed studies in which anon-bacteriophage RNA was encapsidated by MS2 Coat Protein. Theirapparent goal was to determine if the 21 nucleotide Operator (pac site)would confer MS2-specific packageability to non-bacteriophage RNA invivo. E. coli was co-transformed with two plasmids: one encoding MS2Coat Protein and the other encoding β-galactosidase (lacZ). The lacZgene was modified such that it had the MS2 Operator sequence clonedupstream of it. The E. coli were induced such that the Operator-lacZhybrid RNA was co-expressed with the MS2 Coat Protein. The Coat Proteindimer bound to the Operator, triggering the encapsidation of the lacZRNA to form “virus-like particles”. The virus-like particles werepurified by a CsCl gradient. The buoyant density of these virus-likeparticles had a much greater density distribution than did the wild-typeMS2 bacteriophage. The MS2 banded tightly at 1.45 g/cc whereas thevirus-like particles ranged in density from 1.3 to 1.45 g/cc, suggestingsubstantial heterogeneity in the RNA content of the virus-likeparticles. In other words, the Pickett and Peabody virus-like particleswere packaging different lengths of RNA and/or different species ofRNAs.

The results of the Pickett and Peabody work were not as expected. ThelacZ RNA purified from these virus-like particles was degraded to amajor species of ˜500 bases as opposed to the expected full length 3000bases. This 500 base RNA was only detectable by the sensitive Northernblotting procedure. The authors did not know if the degradation occurredbefore or after encapsidation, but suggested that these viral-likeparticles may be sensitive to ribonuclease digestion. It was found thatthe majority of the RNA packaged was actually 2 species, 1800 bases and200 bases in size. These two RNA fragments were easily detected aftergel electrophoresis and methylene blue staining. The 500 baseOperator-lacZ RNA fragment was not visible by methylene blue staining.It was only detected by Northern blotting using a lacZ probe. Theseauthors concluded that the 0.2 and 1.8 kb RNAs were derived from E. colipre-16S rRNA. The host E. coli RNA was packaged in preference to theOperator-lacZ RNA indicating that the specificity of the Pickett andPeabody bacteriophage packaging system was poor.

In other studies, Pickett and Peabody modified the packaging of theOperator-lacZ RNA by changing the ratios of the Coat Protein andOperator-lacZ RNA produced in E. coli. By increasing the concentrationof the Operator-lacZ RNA and decreasing the concentration of the CoatProtein, they were able to encapsidate mainly the Operator-lacZ RNA andno detectable pre-16S rRNA. These results suggested that the originalPickett and Peabody packaging strategy suffered in specificity becausethey were unable to reach and maintain the appropriate molar ratio ofCoat Protein to Operator-lacZ RNA optimal for packaging the target RNA.Even in the second set of packaging studies, the concentrations of theCoat Protein and Operator-lacZ RNA were only coarsely adjusted. ThePickett and Peabody system had no feedback mechanism to maintain theoptimal ratio of Coat Protein to Operator-lacZ RNA for packaging.

In their second set of packaging studies, Pickett and Peabody did notcharacterize the RNA that was packaged with the modified procedure. TheRNA was not purified from the virus-like particles and assessed by, forexample, gel electrophoresis. Furthermore, the virus-like particles inthis study or the previous study were not characterized for theirability to protect the encapsidated lacZ RNA from ribonucleases. Therewas no discussion as to the yield of virus-like particles orOperator-lacZ RNA obtained from the Pickett and Peabody studies.

Currently, there are two major methods for the synthesis of RNA speciesof a specific sequence: chemical synthesis and in vitro transcription.Although the chemical synthesis of RNA can produce very pure product, itis both expensive and it is limited to synthesizing oligonucleotides notmuch longer than 30 bases. Chemical synthesis is most suitable forgenerating antisense RNA oligonucleotides, which are generally 15 to 30bases in length. However, most of the applications for RNA, such asprobe synthesis and in vitro and in vivo translation, require longer RNAproducts, in the range of 100 bases to several kilobases.

RNA synthesis by in vitro transcription became a practical method asdeveloped by Melton et al. (1984). The RNA polymerase from bacteriophageSP6 was used to transcribe DNA templates containing an SP6 bacteriophagepromoter. Since then, promoter/polymerase systems have been developedfor the T7 and T3 bacteriophages as well. These in vitro systems requirea bacteriophage RNA polymerase, a DNA template encoding a phage promoterupstream of the sequence to be transcribed, an appropriate buffer andribonucleotides. Each of these components must be ultrapure and free ofribonucleases to prevent degradation of the RNA product once it istranscribed. Conditions have been optimized which generate 100 to 150 μgof RNA from 1 μg of template (MEGAscript U.S. Pat. No. 5,256,555).Although in vitro transcription is currently the best method ofsynthesizing long RNA sequences, it is expensive for very large scaleproduction in terms of gram quantities of product due to the largequantities of ultrapure enzymes, nucleotides and buffers needed. Yet,such large quantities of RNA are needed for example in vaccination orgene therapy where transient gene expression is desired.

RNA bacteriophage capsids have been assembled in vitro to act as a drugdelivery system, called Synthetic Virions (Stockley, 1994). The OperatorRNA is synthesized chemically and then conjugated to therapeuticoligonucleotides (antisense DNA/RNA or ribozymes) or cytotoxic agents(ricin A chain). The conjugated Operator is then mixed with Coat Proteinin vitro to trigger specific encapsidation of the non-phage molecules.The Synthetic Virions are conjugated to ligands which promote uptake incells by receptor mediated endocytosis. Once inside the cell, theSynthetic Virions disassemble and release the therapeutic molecule. Thedisassembly of the Synthetic Virion is facilitated by the low pH of theendosomal compartments.

RNA has been used to transfect cells in vitro and in vivo to producetransient expression of the encoded protein. One of the applications forRNA transfection is cancer vaccination (Conry, 1995). RNA has theadvantage that expression is transient due to its lability and it is notable to integrate into the host's genome. The use of DNA for nucleicacid vaccination with oncogenes could possibly induce neoplasms. The DNAcould integrate into the host genome, leading to a malignanttransformation. DNA encoding an oncogene may replicate within cells overperiods of months leading to the expression of the oncogene product overthe same time period. It has been demonstrated that prolonged expressionof some oncogenes in cells may result in their transformation. Thecurrent methods for delivering RNA into cells is either injecting nakedRNA, cells from tissue culture mixed with naked RNA, or RNA complexedwith cationic liposomes into the tissue of an animal (Lu, 1994; Dwarki,1993). A problem with these delivery systems is the susceptibility ofthe RNA to ribonucleases in the tissue culture medium or in theextracellular fluids of the host. Transfection efficiency diminishes ifthe mRNA is degraded before it can reach its target. Transfectionefficiency would be improved by increasing the half-life of the RNAprior to its entry into the cell.

SUMMARY OF THE INVENTION

The present invention contemplates various aspects and uses of nucleaseresistant nucleic acids. The invention contemplates various methods ofmaking such nuclease resistant nucleic acids. The invention contemplatesthe use of viral-like systems to produce large amounts of nucleic acid.In preferred embodiments, this nucleic acid is ribonuclease resistant.The invention contemplates the use of nuclease resistant nucleic acids,particularly ribonuclease resistant nucleic acids, in various diagnosticassays.

A primary aspect of the invention is the preparation and use of nucleaseresistant nucleic acid standards. Internal standards play an importantrole in confirming test results. They also provide a means forquantification. The detection and quantification of specific RNAs insamples has become prevalent with the advent of RT-PCR™. The internalstandard for RT-PCR™ studies should be an RNA molecule, as it controlsfor both the reverse transcription and PCR™ amplification steps. This isproblematic, as RNA is particularly susceptible to RNase degradation.Altered test results could be produced by partial or completedegradation of an RNA standard either during storage or afterintroduction to a sample. The likelihood of at least partial RNAdegradation is quite high, given that many of the RNA detection schemesare designed to detect viral RNAs in serum samples, where relativelyhigh quantities of various RNases are located. The ideal internalstandard for RNA diagnostic assays is a molecule that is functionallyequivalent to RNA in the assay format, but resistant to degradation bynucleases. Three general methods can be imagined for protecting RNA fromenzyme-mediated degradation in an environment in which RNases areactive: (1) microencapsulating the RNA inside an impenetrable structure,(2) non-covalently binding the RNA with molecules that deny access ofnucleases to the standard, and (3) chemically altering the structure ofthe RNA in such a way that it is no longer a substrate for nucleaseswhile still being functionally equivalent to RNA in the assay format. Amore detailed description, examples, and enablements for each areprovided below.

The nucleic acids in the standards of the invention can be used inquantifying assays. These standards may be used for a variety ofpurposes such as quantitative RNA standards (to determine the absolutecopy number of a specific RNA sequence), specifically to quantify thenumber of RNA viruses such as HIV-1, HIV-2, HCV, HTLV-1, HTLV-2,hepatitis G, enterovirus, dengue fever virus, or rabies, in plasma,serum, or spinal fluid. They may also be used to quantify the expressionof specific mRNA in cells or tissue by an RT-PCR™ assay. The standardsmay be internal or external. An internal standard is mixed with thesample at a known concentration such that the sample and the standardare processed and assayed as one. Thus, differences in the efficiency ofthe s assay from sample to sample are normalized using the signalgenerated by the internal standard. An external standard is processedand assayed at a known concentration in parallel with the sample but itis processed separately from the sample. Several differentconcentrations of the external standard may be processed simultaneouslyto produce a standard curve which may then be used to determine thevalue of the unknown sample. Internal and external standards may both beused for quantification but internal standards are generally regarded asmore accurate. The standards may be used as qualitative standards actingas positive controls in diagnostics, for example, bacterial, fungal, orparasitic diseases which are diagnostics RNA based or in RT-PCR™ assaysto indicate that all of the reagents are functioning properly. Thesestandards may be used to measure the integrity of an RNA isolationprocedure by measuring the amount of degradation observed in theprotected RNA after it has been subjected to the isolation procedurefollowed by Northern blotting. They may be used as environmental tracersto follow the flow of groundwater or to label the waste of individualcompanies with a unique nucleic acid sequence which can be traced backto the offending company.

The present invention is particularly useful for viral quantification.There are many new nucleic acid based assays in the process of beingdeveloped and/or marketed, i.e., Roche Diagnostic Systems, Amplicor™ HIVMonitor™ and Amplicor™ HCV Monitor tests; Organon Teknika, NASBA HIVkit; GenProbe, Transcription Mediated Amplification HIV kit; and ChironCorp., branched DNA (bDNA) signal amplification assay for HIV and HCV.These assays detect pathogenic human viruses such as HIV and HCV inhuman plasma or serum. These assays are highly sensitive, detecting evenless than 300 virions per 1.0 ml of plasma. In their current format,several of these nucleic acid based assays use naked RNA for theirquantitative standards. Unfortunately, these naked RNA standards arevery susceptible to ribonuclease degradation and thus the results of theassay may be compromised.

One primary embodiment of the present invention relates to nucleic acidstandards comprising nuclease resistant recombinant nucleic acidsegments comprising a sequence coding a standard nucleic acid. In somepreferred embodiments, the nucleic acid standard is an RNA standardcomprising a ribonuclease resistant RNA segment comprising a sequencecoding a standard RNA. As used herein the terms “standard nucleic acid”and “standard RNA” refer respectively to nucleic acids and RNAs that aresuitable for use as a standard in the particular assay to be employed.The present invention contemplates a ribonuclease resistant recombinantRNA which is highly suitable as an RNA standard for quantifying RNAviruses, although it need not be recombinant and may be used as an RNAstandard for RNA isolated from any source, such as cells from tissuecultures. In particular, the structure of an RNA bacteriophage may bemodified to package a recombinant RNA (reRNA) molecule. The reRNAsequence serves as an RNA standard for the quantification of aparticular RNA sequence/target.

In regard to the invention, the terms “nuclease resistant” and“ribonuclease resistant” mean that a nucleic acid exhibits some degreeof increased resistance to nuclease over a naked, unmodified nucleicacid of the same sequence.

There are a variety of methods that may be employed to render a nucleicacid segment nuclease resistant. The nucleic acid segment may bechemically modified, coated with a nuclease resistant coating, or cagedin a nuclease resistant structure. For example, the RNA standard can bea chemically modified RNA that is resistant to ribonuclease. Another wayin which to render a recombinant RNA segment ribonuclease resistant isto coat it with a ribonuclease resistant coating. Such a coating can beanything that binds in a sequence dependent or independent manner to theRNA and renders the RNA ribonuclease resistant. In some cases, the RNAstandard is a recombinant RNA that is caged in a ribonuclease resistantstructure. Methods of caging RNA involve the partial encapsidation ofthe RNA in viral proteins, partial lipid encapsulation of the RNA,partially trapping the RNA in polymer matrices, etc.

In some preferred embodiments of the invention, the ribonucleaseresistant structure is comprised of a viral Coat protein that partiallyencapsidates the RNA standard. The RNA is transcribed in vivo in abacterial host and then encapsidated by bacteriophage proteins. This“caging” of the RNA results in RNA which is protected from ribonuclease(Armored RNA™). Although the nucleic acid or RNA may be completely orsubstantially caged in the nuclease resistant structure, partially cagednucleic acids and RNAs are also within the scope of the presentinvention as long as the partial caging renders the nucleic acid or RNAnuclease or ribonuclease resistant. Thus, when used herein the terms“encapsidation,” “encapsulation,” “trapped,” etc. encompass structureswherein the encapsidation, encapsulation, trapping etc. is partial aswell as substantial or substantially complete so long as the resultantstructure is nuclease or ribonuclease resistant as those terms are usedherein.

In a specific preferred embodiment, the invention relates to aribonuclease resistant recombinant RNA (“reRNA”) standard. These ArmoredRNA™ (AR) standards are ribonuclease resistant due to the encapsidationof the reRNA by bacteriophage proteins. The intact RNA is easilyextracted from the Armored RNA™ standard particles by common RNAextraction methods such as the guanidinium and phenol method(Chomczynski, 1987). The non-bacteriophage RNA may be used in manyapplications: as an RNA standard for quantification, as RNA sizestandards, and for transient gene expression in vitro and in vivo.

The Armored RNA™ can be calibrated to serve as RNA standards inquantitative assays to determine the absolute number of RNA viruseswithin a plasma sample. The Armored RNA™ can be subjected to extremeribonuclease treatment without any degradation of the RNA standard.Armored RNA™ is very durable and can be stored for an indefinite time at4° C., or even room temperature, in the presence of ribonucleases. Thereis no known RNA standard with these qualities. Armored RNA™ differs inseveral features from prior art virus-like particles such as those ofPickett and Peabody. The bacteriophage sequence of the reRNA of thePickett and Peabody particles consisted only of the Operator sequence(or pac site) which is required for Coat Protein recognition of the RNAto initiate packaging. The Armored RNA™ contains about 1.7 kb ofbacteriophage RNA sequence encoding the Maturase, the Coat Protein andthe pac site. The inclusion of the long stretch of bacteriophagesequence within the packaged reRNA may contribute substantially toforming a macromolecular structure most similar to the wild-type MS2structure. Further, there may be other, as of yet uncharacterized,sequences within the bacteriophage RNA that recognizes Coat Protein andMaturase that contribute to assembling the bacteriophage particle into astructure that protects the packaged RNA. Non-bacteriophage RNAs can bepackaged by Coat Protein alone as demonstrated by Pickett and Peabodybut these non-bacteriophage RNA sequences are not entirely ribonucleaseresistant. Besides maximizing the possibility of assuming the correctbacteriophage structure, the inclusion of the extra bacteriophagesequence in the Armored RNA™, as opposed to the Pickett and Peabodyvirus like particles, also increases the specificity of the RNA to bepackaged by the bacteriophage proteins. The Pickett and Peabody viruslike particles contained mainly the host E. coli pre-rRNA over thetarget RNA unless the ratio of the Coat Protein to reRNA was decreased.

A preferred strategy for synthesizing the Armored RNA™ is one that hasbeen optimized by producing a self-regulating feedback mechanism tomaintain the optimal ratio of Coat Protein to reRNA for assembly. TheCoat Protein is encoded in the reRNA and the reRNA is only available fortranslation in its unassembled form. Thus, when the appropriateconcentration of Coat Protein has been translated from the reRNA, itbegins to package the reRNA. More Coat Protein cannot be translateduntil more reRNA is transcribed from the recombinant plasmid. ThePickett and Peabody strategy lacked a mechanism for maintaining aconstant ratio between these two molecules. Pickett and Peabody used atrans mechanism for packaging the Operator-lacZ RNA. The Coat ProteinRNA was transcribed from a different plasmid and therefore, the CoatProtein was being translated from a different RNA than it was topackage. Since there is no Operator on the Coat Protein RNA, the CoatProtein RNA is continually being transcribed and the Coat Protein iscontinually being translated. After induction, there is no regulation ofthe synthesis of the Coat Protein. Similarly, there is no control of thetranscription of the Operator-lacZ RNA. Thus the transcription of bothRNAs is constitutive and translation of the Coat Protein isconstitutive. In contrast, in some embodiments, the Armored RNA™ methodis a cis method where the Coat Protein is being translated from the sameRNA that is to be packaged. The production of the Coat Protein isregulated at the level of translation because once the concentration ofCoat Protein is high enough, it encapsidates the RNA from which it isbeing translated and thus prevents any further Coat Protein from beingtranslated from that RNA. By this autoregulatory method, the levels ofCoat Protein cannot become so high that RNA is encapsidated in anon-specific fashion.

Armored RNA™ may be produced using minimal bacteriophage sequence thatencodes the binding sites for Maturase and Coat Protein (or even less)while providing the Maturase and Coat Protein in trans. The maximal sizeof RNA that can be encapsidated and remain ribonuclease resistantremains to be defined. However, the wild type MS2 bacteriophage containsan RNA genome of ˜3.6 kb. Since the structure of these bacteriophage isiscosahedral, it is likely that the maximal size will be ˜4 kb. Thus,the potential to replace the sequences encoding the Maturase and theCoat Protein with a foreign sequence relevant to the user, may beadvantageous. One skilled in the art can readily perform a systematicset of studies to determine the minimal amount of bacteriophage sequencenecessary to produce Armored RNA™. One advantage of Armored RNA™ inthese applications is that they are non-replicative and therefore,aberrantly high signals would not be detected due to viral replication.

The stability of Armored RNA™ indicates that the packaged RNA maywithstand extreme environmental conditions. This property may be usefulin using Armored RNA™ as molecular markers to trace the origin ofpollutants. For instance, the Armored RNA™ could be spiked into thewaste containers of different companies. The Armored RNA™ for eachcompany would contain a unique nucleotide sequence which would identifythat company. In the event of a spill, a sample would be taken, RNAwould be isolated and RT-PCR™ performed to determine the unique sequenceof the Armored RNA™ and identify the company responsible for the spill.In a related application, the Armored RNA™ could be used byenvironmentalists to trace the flow of groundwaters.

There are many possible methods of creating genes that, when expressedin vivo, will result in Armored RNA™ compositions in which RNA isprotected against ribonuclease in a viral Coat Protein synthesized invivo.

In order to understand some aspects of the invention, it is necessary tounderstand the components of a bacteriophage, for example, the MS2bacteriophage. The RNA genome is ˜3.6 kb and encodes 4 differentproteins: the Maturase, the Coat Protein, the Lysis Protein and theReplicase. The Coat Protein composes most of the mass of the MS2bacteriophage particle. It is a small protein of ˜14 kD in size butthere are 180 molecules of this protein which encapsidate each moleculeof the bacteriophage RNA genome. In total, the Coat Protein moleculesprovide ˜2,500 kD of the total bacteriophage mass of ˜3,500 kD. There isone molecule of Maturase protein per bacteriophage particle which is ˜44kD in size. The Maturase serves to protect the RNA genome fromribonuclease degradation and it is the receptor for the F pilus for E.coli infection. The Lysis Protein and the Replicase are not a componentof the bacteriophage molecule. The Lysis Protein is involved in lysingthe E. coli cell to release the bacteriophage particles. The Replicaseprotein and 3 other E. coli host proteins compose a protein complexwhich is responsible for replicating the RNA genome and synthesizing alarge number of copies for packaging.

In this application, cis refers to a protein binding to the same RNAtranscript species from which it was translated. Trans refers to aprotein binding to an RNA transcript species other than the RNAtranscript species from which it was translated.

The simplest composition may be Coat Protein and a RNA encoding anon-bacteriophage sequence either with or without encoding one or moreOperator sequences. Another composition may comprise Coat Protein and areRNA encoding one or more Operators and a non-bacteriophage sequence.

Another composition may be Coat Protein and Maturase and a reRNAencoding one or more Operator sequences, one or both Maturase Bindingsites and non-phage sequence. The Coat Protein and the Maturase areprovided in trans.

Another composition may be Coat Protein and a reRNA encoding CoatProtein, one or more Operator sequences and non-phage sequence.Including more than one Operator may serve to endow extra protectionagainst ribonucleases and increase the specificity of the Coat Proteinfor the reRNA over host RNA.

Another composition may be Coat Protein and Maturase and a reRNAencoding Coat Protein, Operator sequence, Maturase Binding site andnon-phage sequence. The Maturase protein is provided in trans.

Another composition may be Coat Protein and Maturase and a reRNAencoding Coat Protein, Maturase (which includes a Maturase bindingsites), one or more Operator sequences and non-phage sequence.

Another composition may be Coat Protein and Maturase and a reRNAencoding Coat Protein, Maturase (which includes a Maturase bindingsites), one or more Operator sequences, the Maturase Binding Sitelocated at the 3′ end of the MS2 genome and non-phage sequence.

Another composition may be Coat Protein and Maturase and a reRNAencoding Coat Protein, Maturase, one or more Operator sequences, most ofthe C-terminal coding region of the Replicase (so that none of theprotein is synthesized in vivo). Further, this composition may comprisea sequence coding the entire active Replicase such that the Replicasewill function.

Another composition may be Coat Protein and Maturase and a reRNAencoding Coat Protein, Maturase, one or more Operator sequences, most ofthe C-terminal coding region of the Replicase (so that none of theprotein is synthesized in vivo) and the Maturase Binding Site located atthe 3′ end of the MS2 genome and non-phage sequence.

In some embodiments, there will be no need to have an Operator sequenceto package RNA. The co-expression of Coat Protein and RNA can lead tooperator-less RNA being packaged and protected in a manner that may beless specific than produced when an Operator sequence is present.

Other compositions may comprise any of the above, in conjunction withthe Lysis Protein. The Lysis Protein may be provided in either.

In each of these compositions, the non-phage RNA may be positioned at avariety different regions of the reRNA. For example, in the firstcomposition, the reRNA may encode 2 Operators and non-phage RNA. BothOperators may be located 5′ or 3′ of the non-phage RNA or there may beone Operator at each end of the non-phage RNA. If there is only a singleOperator, it may be preferable to position it at the 3′ end of thefull-length transcript so that only full-length transcripts arepackaged. Including a Maturase Binding Site near the 3′ end may have asimilar advantage towards packaging full length RNA. In the wild-typephage genome, the Maturase Binding Sites are located within the Maturasecoding sequence and at the 3′ end of the genome. In the compositionswhere the Coat Protein is provided in trans, it is preferable that thereis no Operator sequence encoded on the same RNA transcript as the CoatProtein or the Coat Protein may bind both the Coat Protein RNAtranscript and the non-phage-Operator RNA, producing a mixed populationof capsids.

Using more than one Operator sequence per RNA molecule may be expectedto increase the specificity of the Coat Protein for the target RNA anddecrease the possibility of packaging host RNA in vivo as in the Pickettand Peabody studies. In vitro studies investigating the binding kineticsof Coat Protein with the Operator sequence demonstrated that CoatProtein bound in a cooperative manner to an RNA molecule encoding twoOperator sequences and that the Coat Protein bound to the RNA with twoOperators at a much lower concentration than an RNA with a singleOperator (Witherell, 1990). Increasing the specificity of binding mayalso be accomplished using a mutant Operator sequence with a higheraffinity for Coat Protein than the wild type sequence (Witherell, 1990).

Major aspects of the invention may be summarized as follows. One primaryembodiment of the present invention relates to nucleic acid standardscomprising nuclease resistant nucleic acid segments comprising sequencescoding a standard nucleic acid. In some preferred embodiments, thenucleic acid standard is an RNA standard comprising a ribonucleaseresistant RNA segment comprising a sequence coding a standard RNA.

There are a variety of forms ribonuclease resistant RNA standards thatcan be employed. The RNA can be chemically modified RNA that isresistant to ribonuclease. A chemically modified RNA may be comprised ofchemically modified nucleotides. These nucleotides are modified so thatribonucleases cannot act on the RNA. The chemically modified RNA isprepared by chemical modification of an RNA or a previously transcribedRNA transcript. Alternatively, the chemically modified RNA may betranscribed or synthesized from nucleotides that have already beenchemically modified.

An RNA standard may also comprise an RNA that is bound non-covalently,or coated with, a ribonuclease resistant coating. Such binding, whichmay be sequence dependent or independent, renders the RNA ribonucleaseresistant. In some embodiments, the bound molecule is comprised of aprotein. Examples of such binding proteins are MS2/R17 coat protein,HIV-1 nucleocapsid protein, gp32, the regA protein of T4, or the gp32 ofbacteriophage T4. In other cases, the non-covalently bound molecule iscomprised of a small molecule. For example the polyamines, spermineand/or spermidine. The ribonuclease-resistant coating may also becomprised of a nucleic acid. In some preferred embodiments, the nucleicacid hybridizes to the recombinant RNA, blocks nucleases, and can serveas a primer for reverse transcriptase. In other cases, poly-L-lysine andcationic detergents such as CTAB may be used to coat and protect RNA.

In other embodiments of the invention, the ribonuclease resistant RNAsegment is a caged ribonuclease resistant structure, that is, the RNAsegment is partially encapsulated in a ribonuclease resistant structure.For example, the ribonuclease resistant structure may be comprised oflipids. In some cases, a lipid ribonuclease resistant structure willcomprise a liposome. In other embodiments, the ribonuclease resistantstructure is a synthetic microcapsule, such as a polymer matrix. Someexamples of useful polymer matrices comprise agarose or acrylamide.

In some preferred embodiments, the invention contemplates a nucleic acidstandard comprising a ribonuclease resistant structure comprising astandard nucleic acid segment encapsidated in viral Coat Protein. Apreferred embodiment of the invention contemplates an RNA standardcomprising a ribonuclease resistant RNA segment comprising a sequencecoding a standard RNA. Encapsidation of a RNA segment in a viral CoatProtein can render it resistant to ribonuclease, hence the term ArmoredRNA™.

The viral Coat Protein may be any native or modified viral Coat Protein,but, in many preferred embodiments, the viral Coat Protein is abacteriophage viral Coat Protein. Such bacteriophage viral Coat Proteinsmay be of an E. coli bacteriophage of genetic subclass A or B; in somepreferred embodiments, the bacteriophage viral Coat Protein is of an E.coli bacteriophage of genetic subclass A. A bacteriophage viral CoatProtein can be of an E. coli bacteriophage in serological group I, II,II, or IV, with some preferred embodiments employing a bacteriophageviral Coat Protein from E. coli bacteriophage of serological group I. Incertain specifically preferred embodiments, the bacteriophage viral CoatProtein is of an MS2JR17 bacteriophage. The bacteriophage viral CoatProtein may also be of a Pseudomonas aeruginosa RNA bacteriophage, forexample, the Pseudomonas aeruginosa PRR1 or PP7 bacteriophage. Thebacteriophage viral Coat Protein may further be of a filamentousbacteriophage, and, because such bacteriophage can comprise a longer RNAsegment than many other bacteriophage, this is an embodiment ofparticular interest. It is also contemplated that the bacteriophage ofthe archae bacteria will be useful in the invention (Ackerman, 1992). Ofcourse, the viral Coat Protein need not be from a bacteriophage, and theinvention contemplates viral Coat Proteins from plant or animal virus,for example, tobacco mosaic virus (Hwang 1994a; Hwang, 1994b; Wilson,1995), the alphaviruses (Frolov, 1996), HBV, feline immunodeficiencyvirus, and Rous sarcoma virus will all be useful. The viral Coat Proteinmay be a native or a modified viral Coat Protein. Modified viral CoatProteins may be used to obtain certain desirable characteristics, suchas greater or lesser viral coat resistance. Modified viral Coat Proteinsmay be made by any of a number of methods known to those of skill in theart, including PCR™-based and other forms of site-directed mutagenesis.

In certain preferred embodiments, the ribonuclease resistant RNA segmentis bound to a viral Maturase protein. For example, the RNA standard maycomprise a viral Maturase protein bound to a viral Maturase binding siteon a recombinant RNA segment. The viral Maturase protein and/or viralMaturase protein binding site may be native or modified. Modificationsin the base sequence of the Maturase binding site and in the amino acidsequence of the Maturase may be made by any of a number of methods knownto those of skill. A viral Maturase binding site is found in the RNAsequence that encodes a native Maturase. Therefore, the RNA sequence maycontain within itself an RNA coding for the Maturase. Further, sinceMaturase binding is purported to have some effect on the stability ofRNA segments, it is contemplated that multiple Maturase binding sitesand/or Maturase coding sequences may be included in the RNA segment.

The RNA segment which codes for a standard ribonucleic acid may alsocomprise a sequence coding a Replicase protein, and the Replicaseprotein may or may not be expressed or expressible from that sequence.In certain preferred embodiments, the sequence coding the Replicaseprotein codes a modified Replicase protein that is not active.

The RNA segment will typically comprise an Operator coding sequence,and, in many preferred embodiments, a viral Maturase protein bindingsite which may be included in a viral Maturase protein coding sequence.The RNA segment may further comprise a viral Coat Protein codingsequence of the type discussed above.

There are many embodiments of the RNA segment comprising a sequencecoding a standard RNA, a few examples of which are given below. In somevery basic embodiments, the RNA segment comprises an Operator sequenceand a viral Coat Protein sequence. In other basic embodiments, the RNAsegment comprises an Operator sequence, a viral Coat Protein sequence,and a non-bacteriophage sequence. In other embodiments, the RNA segmentcomprises at least two Operator sequences and a non-bacteriophagesequence. The RNA segment may comprise an Operator sequence, a sequencecoding a viral Maturase protein, and a non-bacteriophage sequence.Further, in some preferred embodiments, the RNA segment comprises anOperator sequence, a sequence coding a viral Maturase protein, asequence coding a viral Coat Protein and a non-bacteriophage sequence.The RNA segment may comprise an Operator sequence, at least two viralMaturase binding sites, a sequence coding a viral Maturase protein, asequence coding a viral Coat Protein and a non-bacteriophage sequence.Alternatively, the RNA segment may comprise an Operator sequence, atleast two viral Maturase binding sites, a sequence coding a viralMaturase protein, a sequence coding a viral Coat Protein, anon-bacteriophage sequence, and a sequence coding a Replicase protein.The RNA may comprise all or part of the recombinant RNA segment codedfor in the sequence of pAR-1 or pAR-2.

In some preferred embodiments, the RNA segment comprises a bacteriophagesequence from an RNA bacteriophage and a non-bacteriophage sequence. Thenon-bacteriophage sequence may be inserted into a multiple cloning site.The non-bacteriophage sequence may be a viral, bacterial, fungal,animal, plant, or other sequence, although, in certain preferredembodiments it is a viral sequence. Multiple Operators may be on eitherterminus of the non-bacteriophage sequence, or may flank the sequence.Multiple Operator sequences may be useful for packaging largernon-bacteriophage sequences.

The non-bacteriophage sequence is often a sequence adapted for use as astandard in detection and/or quantification of an RNA by, for example,PCR™-based procedures. In specific embodiments, the non-bacteriophagesequence is a sequence adapted for use in detection and/orquantification of an RNA of diagnostic value. For example, thenon-bacteriophage sequence can be a sequence adapted for use as astandard in detection and/or quantification of HIV-1, HIV-2, HCV,HTLV-1, HTLV-2, hepatitis G, an enterovirus or a blood-borne pathogen.In some particularly interesting embodiments, the non-bacteriophagesequence is adapted for use in the detection of such viral diseases asHIV-1, HIV-2, HCV, HTLV-1, or HTLV-2. Adaptation of thenon-bacteriophage sequence can be accomplished in any manner that willrender the sequence suitable for detection and/or quantification of thetested RNA. In some embodiments, the non-bacteriophage sequence adaptedfor use as a standard in detection and/or quantification of an RNA ofinterest by modifying the native RNA sequence to be detected ormonitored so that it is distinguishable from the native sequence. Forexample, detection and/or quantification of HIV-1 can be accomplishedwith a non-bacteriophage sequence comprising a modified HIV-1 sequence.The RNA standard may comprise a non-bacteriophage sequence adapted foruse as a standard in detection and/or quantification of a blood-bornepathogen, such as a plasmodium, trypanosome, Francisella tularensis, orWucheria bancrofti.

The bacteriophage sequence of the RNA standard may be a sequence fromany E. coli bacteriophage of any genetic subclass, for example, subclassA. Further the bacteriophage sequence may be a sequence from an E. colibacteriophage in serological group I, II, II, or IV. In certainembodiments, the bacteriophage sequence is a sequence from an MS2/R17bacteriophage. Of course, the bacteriophage sequence can also be asequence from a Pseudomonas aeruginosa RNA bacteriophage, such as thePRR1 or PP7 bacteriophage, or a filamentous bacteriophage.

Other embodiments of the invention contemplate a RNA segment comprisingvarious of the sequences discussed above. The RNA standard segment maybe encapsidated in viral Coat Protein, or free from viral Coat Protein.For example, a recombinant RNA may be free of viral Coat Protein duringthe RNA standard production process or during an assay after isolationof the recombinant RNA segment from the viral Coat Protein. The RNA maybe of any of the various forms discussed above, and may compriseOperator site(s), Maturase binding site(s), Coat Protein codingsequence(s), Maturase coding sequence(s), non-bacteriophage sequence(s),restriction enzyme sequence(s), active or non-active Replicase codingsequence(s), active or non-active Lysis Protein coding sequence(s)and/or other sequences.

The invention also contemplates DNA vectors adapted for use in thesynthesis of a RNA standard comprising recombinant RNA segmentencapsidated in viral Coat Protein. Such vectors are transfected intocells, for example E. coli, and function to cause the cells to produceRNA encapsidated in viral coat protein. A basic vector may comprise asequence coding an Operator sequence and a viral Coat Protein sequence.Alternatively, the vector may comprise a sequence coding two Operatorsequences and a non-bacteriophage sequence. In some embodiments, thevector may comprise a sequence encoding an Operator sequence, a sequencecoding a viral Maturase binding site, and a multiple cloning site. Themultiple cloning site may be either downstream or upstream of a sequenceencoding a viral Maturase binding site. The vector may further comprisea sequence coding a viral Maturase protein and/or a Maturase bindingsite. The sequence coding the viral Maturase binding site may becomprised within the sequence coding the viral Maturase protein. Certainpreferred embodiments comprise a sequence coding a viral Coat Proteingene, an Operator sequence, and a multiple cloning site. A DNA sequencecoding a non-bacteriophage sequence may be inserted into the multiplecloning site of such a DNA vector, and the non-bacteriophage sequencemay be any of the sequences discussed above.

The invention contemplates collection tubes containing a nucleic acidstandard comprising recombinant nucleic acid encapsidated in viral CoatProtein. Such collection tubes may be adapted for use in collection of abody fluid such as blood, urine, or cerebrospinal fluid. For example,the collection tube may be a vacuum tube for the drawing of blood. Suchcollection tubes can streamline a diagnostic procedure by providing anucleic acid standard in a body fluid sample at the time of drawing ofthe fluid and eliminating the need to add the standard as a part of theassay procedure.

The present invention contemplates methods for assaying for the presenceof a tested nucleic acid in a nucleic acid sample using the nucleic acidstandards described above. The “nucleic acid sample” may also bedescribed herein as a nucleic acid composition. A nucleic acidcomposition, as used herein, is taken to mean any composition, usually aliquid composition that contains one or more nucleic acid molecules orpolymers. The composition may also comprise buffers, salts, solvents, orsolutes and the like, that are derived from a sample along with thenucleic acid composition, or that have been added to the compositionduring or after isolation. Such compositions are typically precipitatedfrom an aqueous solution and resuspended. The nucleic acid standards maybe internal or external standards. Such methods generally comprise thesteps of: (1) obtaining a sample; (2) obtaining a nucleic acid standardcomprising a nuclease resistant nucleic acid segment comprising asequence coding a standard nucleic acid; (3) assaying the sample for thepresence of a tested nucleic acid sequence; and (4) employing thenucleic acid segment comprising a sequence coding a standard nucleicacid as a standard in the assay. The methods may further comprise thestep of isolating a nucleic acid composition from the sample. It iscontemplated that samples may include, but would not be limited toinorganic materials such as a soil sample, any organic material, samplesfrom a plant or animal, and may be tissue samples, or samples of bloodor blood components. This method may further comprise isolating thenuclease resistant nucleic acid segment comprising a sequence coding astandard nucleic acid from a molecule that renders the nucleaseresistant nucleic acid segment comprising a sequence coding a standardnucleic acid nuclease resistant to obtain a nucleic acid segmentcomprising a sequence coding a standard nucleic acid.

Alternatively, this method may further comprise admixing the sample ornucleic acid composition and the nucleic acid standard comprising anuclease resistant nucleic acid segment comprising a sequence coding astandard nucleic acid prior to assaying for the presence of the testednucleic acid sequence. This alternative method may further compriseisolating the nuclease resistant nucleic acid segment comprising asequence coding a standard nucleic acid from a molecule that renders thenucleic acid segment comprising a sequence coding a standard nucleicacid nuclease resistant to obtain a nucleic acid segment comprising asequence coding a standard nucleic acid. Additionally, the sample andthe nucleic acid standard may be admixed prior to isolation of a nucleicacid acid composition from the sample and isolation of the nucleic acidsegment comprising a sequence coding a standard nucleic acid so thatisolation of the nucleic acid composition from the sample and thenucleic acid segment comprising a sequence coding a standard nucleicacid is performed in the same isolation procedure. This streamlines theprocedure and assures that any tested nucleic acid and the nucleic acidstandard are processed in parallel in the same reaction. Such parallelprocessing eliminates many variables that could compromise the resultsof the assay.

The assay may be any that would employ a nucleic acid standard, althoughmany preferred embodiments comprise PCR™ analysis. One of the advantagesof the nucleic acid standards of the invention is that they allow forquantitative assays, such as quantitative RT-PCR™. In RT-PCR™procedures, the nucleic acid segment is typically an RNA comprising asequence coding a standard RNA. Typically quantitative assays willcomprise comparing an amount of tested RNA PCR™ product with an amountof standard RNA PCR™ product. RT-PCR™ analysis will usually comprise:(1) employing a reverse transcription procedure; (2) amplifying anucleic acid sequence and generating a PCRT product; and (3) detectingPCR™ product. In certain embodiments, the amplification step involvesco-amplification of any tested RNA PCR™ product with standard RNA PCR™product. Such co-amplification can be achieved via the use of a singleprimer set adapted for amplification of both tested RNA PCR™ product andstandard RNA PCR™ product from an RT-PCR™ procedure.

The nucleic acid standards may be of any composition described eitherexplicitly or implicitly above. For example, where ribonucleic acidstandards are employed any form of ribonuclease resistant RNA segmentcomprising a sequence coding a standard RNA may be employed, including,but not limited to those involving chemical modification, ribonucleaseresistant coating, or ribonuclease resistant caging.

The sequence coding the RNA standard may comprise a non-bacteriophagesequence, such as a viral sequence. The non-bacteriophage sequence maygenerally be a sequence adapted for use as a standard in detectionand/or quantification of an RNA. In some preferred embodiments, theassay may be employed to detect and/or quantify viral loads in infectionwith HIV-1, HIV-2, HCV, HTLV-1, HTLV-2, hepatitis G, an enterovirus, ora blood-borne pathogen. Presently more preferred embodiments contemplatethe detection and/or quantification of HIV-1, HIV-2, or HCV using an RNAstandard comprising a recombinant RNA with a modified HIV-1, HIV-2, orHCV sequence.

One specific method of the invention contemplates assaying for thepresence of an RNA of diagnostic value by a method comprising: (1)obtaining a sample to be assayed; (2) obtaining an RNA standardcomprising a sequence coding a standard RNA encapsidated in abacteriophage Coat Protein; (3) admixing the sample with the RNAstandard; (4) isolating RNA from the admixture; and (4) assaying for thepresence of the RNA of diagnostic value with a RT-PCR™ analysis.

The invention contemplates methods of making a nucleic acid standardcomprising a recombinant nucleic acid segment encapsidated in viral CoatProtein comprising: (1) obtaining a vector comprising a nucleic acidsequence coding a recombinant nucleic acid segment comprising a sequencecoding an Operator sequence, and a non-bacteriophage sequence; (2)transfecting the vector into a cell; (3) providing a viral Coat Protein;and (4) culturing the cell under conditions allowing for transcriptionof the recombinant nucleic acid segment and encapsidation of therecombinant nucleic acid segment in viral Coat Protein. The recombinantnucleic acid segment may be RNA or DNA. The nucleic acid standard may bepurified from the cells in which it is expressed by any of a number ofmanners known to those of skill for the separation of viral particlesfrom cells. The cell may be any form of cell, although typically abacterial cell, such as E. coli is employed.

Particularly preferred are methods of making RNA standards comprising arecombinant RNA segment encapsidated in viral Coat Protein, whichmethods comprise: (1) obtaining a vector comprising a DNA sequencecoding a recombinant RNA segment comprising a sequence coding anOperator sequence and a non-bacteriophage sequence; (2) transfecting thevector into a cell; (3) providing a viral Coat Protein; and (4)culturing the cell under conditions allowing for transcription of therecombinant RNA segment and encapsidation of the recombinant RNA segmentin viral Coat Protein. In many preferred embodiments, the recombinantRNA will comprise a Maturase binding sequence.

The provision of the viral Coat Protein can be by any number of means.For example, the protein can be expressed separately from thetranscription of the recombinant RNA segment and added into the culturemedium in a concentration such that the recombinant RNA becomesencapsidated once transcribed. However, in most preferred embodiments,the provision of the Coat Protein comprises: (1) obtaining a nucleicacid segment coding a viral Coat Protein; (2) transfecting the nucleicacid segment coding the viral Coat Protein into the cell; and (3)culturing the cell under conditions allowing for expression of the viralCoat Protein. In this embodiment, the nucleic acid segment coding theviral Coat Protein may be a DNA sequence comprised in the vectorcomprising the DNA sequence coding the recombinant RNA segment. The DNAsequence coding the viral Coat Protein may be located cis to the DNAsequence coding the recombinant RNA segment. Further, the DNA sequencecoding the viral Coat Protein can be located in the DNA sequence codingthe recombinant RNA segment. Alternatively, the DNA sequence coding theviral Coat Protein may be located trans to the DNA sequence coding therecombinant RNA segment, although this is not typical of preferredembodiments.

The method of making an RNA standard may comprise the further step ofproviding a viral Maturase protein. The provision of the viral Maturaseprotein can be by any number of means. For example, the protein can beexpressed separately from the transcription of the recombinant RNAsegment and added into the culture medium in a concentration such thatthe recombinant RNA becomes encapsidated once transcribed. However, inmost preferred embodiments, the provision of the Maturase proteincomprises: (1) obtaining a nucleic acid segment coding a viral Maturaseprotein; (2) transfecting the nucleic acid segment coding the viralMaturase protein into the cell; and (3) culturing the cell underconditions allowing for expression of the viral Maturase protein. Inthis case, the nucleic acid segment coding the viral Maturase proteinmay be a DNA sequence comprised in the vector comprising the DNAsequence coding the recombinant RNA segment. The DNA sequence coding theviral Maturase protein may be located cis to the DNA sequence coding therecombinant RNA segment. Further, the DNA sequence coding the viralMaturase protein can be located in the DNA sequence coding therecombinant RNA segment. Alternatively, the DNA sequence coding theviral Maturase protein may be located trans to the DNA sequence codingthe recombinant RNA segment, although this is not typical of preferredembodiments. The recombinant RNA sequence, may, of course, be any ofthose discussed or suggested explicitly or implicitly above.

A preferred embodiment of the method of making an RNA standardcomprising a recombinant RNA segment encapsidated in viral Coat Proteincomprises: (1) obtaining a vector comprising a DNA sequence coding arecombinant RNA segment comprising a sequence coding an Operatorsequence, a sequence coding a viral Maturase binding site, and anon-bacteriophage sequence; (2) transfecting the vector into a cell; (3)obtaining a DNA segment coding a viral Coat Protein and transfecting thenucleic acid segment coding the viral Coat Protein into the cell; (4)obtaining a DNA segment coding a viral Maturase protein and transfectingthe nucleic acid segment coding the viral Maturase protein into thecell; and (5) culturing the cell under conditions allowing fortranscription of the recombinant RNA segment, expression of the viralCoat Protein and the viral Maturase protein, and encapsidation of therecombinant RNA segment in viral Coat Protein. In preferred embodimentsof this aspect of the invention, the DNA segment coding the viral CoatProtein is comprised in the vector comprising the DNA sequence codingthe recombinant RNA segment. More preferably, the DNA sequence codingthe viral Coat Protein is located cis to the DNA sequence coding therecombinant RNA segment. In preferred embodiments of this invention, theDNA segment coding the viral Maturase protein is comprised in the vectorcomprising the DNA sequence coding the recombinant RNA segment and, morepreferably, located cis to the DNA sequence coding the recombinant RNAsegment.

The invention also contemplates methods of making RNA in vivocomprising: (1) obtaining a vector comprising a DNA sequence coding arecombinant RNA segment comprising a sequence coding an Operatorsequence, a sequence coding a viral Maturase binding site, and anon-bacteriophage sequence; (2) transfecting the vector into a cell; (3)obtaining a DNA segment coding a viral Coat Protein and transfecting thenucleic acid segment coding the viral Coat Protein into the cell; (4)obtaining a DNA segment coding a viral Maturase protein and transfectingthe nucleic acid segment coding the viral Maturase protein into thecell; and (5) culturing the cell under conditions allowing fortranscription of the recombinant RNA segment, expression of the viralCoat Protein and the viral Maturase protein, and encapsidation of therecombinant RNA segment in viral Coat Protein. These methods may furthercomprise the step of isolating the recombinant RNA segment from the CoatProtein, and this allows for the production of a large amount of desiredRNA in vivo, i.e., within bacterial cells. The isolated RNA segment maythen be treated to obtain an RNA segment comprising thenon-bacteriophage sequence. For example ribozyme sequences, RNase H anda complementary DNA oligonucleotide that function to generate sequencespecific cuts in the RNA, or other molecular biology tools may be usedto excise undesired RNA from the non-bacteriophage sequence, or aportion thereof. The desired RNA segment may then be purified by meansknown in the art. The DNA vectors, RNA segments, cells, etc. employedand obtained in this method of in vivo transcription may be any of thosedescribed above.

The invention also contemplates methods of encapsidating a RNA segmentin viral Coat Protein in vitro comprising: (1) obtaining a RNA segmentcomprising a sequence coding a standard RNA; (2) obtaining viral CoatProtein; and (3) placing the RNA segment comprising a sequence coding astandard RNA and the viral Coat Protein together under conditionscausing the RNA segment comprising a sequence coding a standard RNA tobecome encapsidated in the viral Coat Protein.

The invention further contemplates methods of delivering RNA to cells invitro or in vivo comprising: (1) obtaining an Armored RNA™ comprising aRNA segment comprising a sequence coding a standard RNA encapsidated inviral coat protein; (2) placing the Armored RNA™ culture with a cell;and (3) culturing the cell under conditions that cause the Armored RNA™to be taken into the cell.

In such a method, the Armored RNA™ may comprise a bacteriophage proteinthat has been modified to facilitate delivery of RNA to a cell. Forinstance, the modified bacteriophage protein is a viral Coat Protein ora Maturase protein.

There are many different single and double stranded DNA bacteriophageswhich infect E. coli and other bacteria. Examples of single stranded DNAbacteriophage include φX174 and M13. Examples of double stranded DNAbacteriophage include T4, T7, lambda (λ), and phage P2. M13 and λ havebeen used extensively by molecular biologists and it is rather simple tocreate recombinants of these bacteriophage. As with the RNAbacteriophage, recombinants of the DNA bacteriophage could beconstructed and quantified with specific DNA sequences to act asquantitative standards for particular DNA viruses using nucleic acidbased assays. Some of the human DNA viruses are HSV, EBV, CMV, HBV,Parvoviruses, and HHV6. The benefit of these standards is that the DNAstandards would be protected against DNases.

RNA synthesized by in vitro transcription may be packaged withbacteriophage proteins in vitro. This method would be useful towardsprotecting RNA species of very specific sequences, that is, the reRNAwould not need to encode the Coat Protein and Maturase sequences. Onlythe binding sequences for Coat Protein and/or Maturase would be includedwithin the RNA transcript and Coat Protein and/or Maturase would beprovided exogenously. Encapsidation may occur co- orpost-transcriptionally. It has been demonstrated that by combining RNAand Coat Protein under the appropriate conditions, the RNA will beencapsidated with Coat Protein to form a phage like particle (LeCuyer,1995). Capsid formation by Coat Protein is stimulated by Operatorsequence or long RNA transcripts (Beckett, 1988). Capsids will formwithout Operator sequence or any RNA but then the concentration of theCoat Protein must be much higher. Therefore, the Coat Protein may bestored at a concentration which does not lead to capsid formation unlessit is added to RNA. Using this strategy, the RNA may or may not requirethe Operator sequence, depending on the length and concentration of theRNA. This strategy may lend itself to packaging mRNA (RNA having a 5′cap and 3′ polyA tail) which may then be used in transfection studies(see below). The Maturase may be required to form structures in whichthe packaged RNA is protected against ribonucleases.

RNA of different discrete lengths may be produced as Armored RNA™ inlarge quantities. The sizes could be mixed in equal mass amounts intheir Armored RNA™ form. This mix may be heated in a denaturing solutionand run on a denaturing formaldehyde agarose gel directly or the RNA maybe purified from the mix and then run on a denaturing formaldehydeagarose gel as RNA size standards. Alternatively, chemically modifiedRNAs of different lengths may be transcribed which are ribonucleaseresistant. These RNAs may be used as size standards in gelelectrophoresis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Consistency of the signal produced by the AR-2 standard whichwas stored at 4° C. AR-2 standard (80 QS RNA equivalents) was added to0.2 ml of HIV positive serum and processed using the Amplicor™ HIVMonitor™ test. Twenty one assays were performed over a 38 day periodusing the same stock of AR-2 standard stored at 4° C. in water.

FIG. 2. The effect incubating intact AR-2 standards or naked AR-2 RNA inhuman serum. A defined amount of RNA either encapsidated as AR-2 (500RNA equivalents), or as naked RNA isolated from AR-2 (5000 RNAequivalents) were incubated with human serum for increasing periods atroom temperature (21° C.). As controls, the naked AR-2 RNA and theintact AR-2 standard were both incubated in parallel in Tris-buffer.Percent recovery is calculated as the OD₄₅₀ X dilution factor (DF) ofthe samples at time zero divided by OD₄₅₀ X DF at the end of eachincubation multiplied by 100.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following examples describe the production and use of ribonucleaseresistant RNA, illustrate the degree to which the reRNA can be protectedfrom ribonuclease activity, and applications of the invention.

EXAMPLE I Production of Armored RNA™ and Use of Armored RNA™ to QuantifyHIV

1. Construction Of Armored RNA™.

The Armored RNA™ standard is a modified version of the RNA bacteriophageMS2 which contains only the Maturase and Coat Protein genes. Afull-length cDNA clone of the RNA bacteriophage MS2 (pMS27) contains allthe genes necessary to produce wild-type, infectious MS2 bacteriophage(Shaklee, 1990).

A fragment of DNA was synthesized from pMS27 by PCR™ encoding theMaturase and the Coat Protein of MS2 using the primers 5′CCTTTCGGGGTCCTGCTCAACTT 3′ (sense primer SEQ ID NO: 1) and 5′GATTAGATCTGAGTTGAACTTCTTTGTTGTCTTC 3′ (antisense primer SEQ ID NO: 2). ABglII restriction sequence was incorporated into the antisense primer tomediate the cloning of this PCR™ product into the expression vectorpSE380 (Invitrogen Corporation). The PCR™ product was purified using theGeniePrep™ (Ambion, Inc.) and eluted from the glass fiber pad with 70 μlof water. GeniePrep™ is a DNA isolation kit that purifies plasmid DNAbased on the common alkaline lysis procedure (Bimboim, 1983). The PCR™fragment was digested with Nco I and Bgl II. Ten μl of 10× React 3Buffer (BRL; 0.5M Tris[pH 8.0]; 0.1M MgCl₂; 1M NaCl) 20 μl water and 3μl of Nco I and Bgl II were added to the purified PCR™ product andincubated 37° C., 2.5 h. An Nco I site is a naturally occurring sequencewithin the MS2 genome located just 5′ of the start codon for theMaturase gene. The digested PCR™ product was again purified withGeniePrep™ (Ambion, Inc.) and eluted with 70 μl of water.

The digested PCR™ fragment was ligated into the Nco I and Bgl II sitesof vector pSE380. pSE380 previously digested with Bgl II and Nco I wascombined with the Bgl II and Nco I digested PCR™ product in a 1×Ligation Buffer (50 mM Tris [pH 7.8]; 10 mM MgCl₂; 10 mM DTT; 1 mM ATP;0.025 mg/ml acetylated BSA, Ambion, Inc.) with T4 DNA Ligase (Ambion,Inc.). The ligation reaction was incubated at room temperature (˜21°C.), 4 h. Ligated product was transformed into competent E. coli strainDH5α cells and then spread onto Cb₁₀₀-LB plates, 37° C., 16 h. Some ofthe resulting colonies were picked, grown in Cb₂₅-LB medium, the plasmidDNA isolated using GeniePrep™ (Ambion, Inc.) and then screened for a DNAinsert with the same size as the PCR™ product by digesting the DNA withNco I and Bgl II. The pSE380 vector contains the E. coli RNA Polymeraseterminator sequence rrnBT₁T₂, 3′ of the multiple cloning site, positions709 to 866.

The resulting construct is named pAR-1. SEQ ID NO:3. Both bacteriophagegenes are downstream of the strong trc promoter which is regulated bythe lacI protein expressed by pSE380. The Operator sequence (pac site)is present in the Nco I/Bgl II fragment just downstream of the CoatProtein sequence. A truncated version of the Lysis gene is also presentbut the peptide encoded is not an active form of the protein. pSE380encodes lac I^(q), the superrepressor, which is inactivated in thepresence of IPTG. Thus, transcription of the bacteriophage genes isdown-regulated until IPTG is added to the culture medium and the trcpromoter is activated. Transcription is terminated by the rrnBT₁T₂terminator sequence.

The Maturase protein, the Coat Protein and the RNA which encoded theseproteins were used for the production of the stable, bacteriophage-likeArmored RNA™ particle. The Maturase protein is hypothesized to be animportant component of Armored RNA™ that is hypothesized to stabilizethe bacteriophage-like particle and endow additional protection for theRNA contained within the bacteriophage like particle. Therefore, manypreferred embodiments of the invention will include the Maturaseprotein. There is a binding site for Maturase protein within theMaturase coding sequence (Shiba, 1981). The Maturase binding site washypothesized necessary to be included in the reRNA as sequence whichcontributed to the packaging of the reRNA. The Coat Protein is animportant component because it makes up the bulk of the bacteriophageparticle. It was hypothesized unnecessary to include the Lysis gene orthe Replicase gene because neither gene is involved in packaging. TheReplicase is not is needed because E. coli RNA Polymerase transcribesfrom plasmid DNA the RNA sense (+) strand encoding Maturase and CoatProtein once transcription is induced. However, as mentioned previously,it may not be necessary to have the genes for the Maturase and CoatProtein in cis with the RNA standard. They might be supplied in transfrom another vector or even incorporated into the E. coli chromosome.

Armored RNA™ was produced using the pAR-1 recombinant plasmid. E. colistrain DH5( harboring this plasmid were grown overnight, 200 rpm, 37° C.in LB medium (10 g/liter tryptone, 5 g/liter yeast extract, 10 g/literNaCl in water, pH 7.0-7.5) with 25 μg/ml carbenicillin (Cb₂₅). 0.2 ml ofthe overnight culture was used to inoculate 2 ml of fresh Cb₂₅-LB mediumand incubated 1.5 h, 37° C., 200 rpm. Expression was induced by addingIPTG to 1 mM and incubating 3 h, 37° C., 200 rpm.

The cells were pelleted and then resuspended in 0.25 ml of 5 mM MgSO₄:0.1 M NaCl: 50 mM Tris (pH 8.0) (Sonication Buffer). The cells weresonicated (Branson Sonifier 450) with the small sonication probe 50%duty cycle, unit 5 power for 5 pulses of the probe. The sonicate wasiced 1 min and then the sonication step was repeated. The sonicate wascentrifuged to pellet the cell debris. 20 μl of supernatant wasincubated with 100 units of E. coli RNase 1 and 2 units of bovinepancreatic DNase 1, 37° C., 40 min, to eliminate E. coli RNA and DNA.After nuclease treatment, 15 μl of supernatant was electrophoresed on anagarose gel in TBE buffer and stained with Ethidium Bromide to assay forArmored RNA™.

The Armored RNA™ had a mobility of a 900-1000 base pair double strandedDNA fragment as compared to two different DNA size standards (lambda DNAdigested with HindIII and pUC19 plasmid DNA digested with Sau 3A) whichwere also run on the gel with the Armored RNA™. The mobility was verysimilar to the wild-type MS2 bacteriophage mobility. Treatment ofArmored RNA™ with DNase or RNase did not affect their intensity ofstaining or their electrophoretic mobility. Ribonuclease 1 (100 units;Ambion, Inc.) and DNase 1 (2 units; Ambion, Inc.) were added separatelyor together to 20 μl of the Armored RNA™ supernatant. The supernatantwas incubated at 37° C. with these enzymes, 40 minutes and thenfractionated on an 0.8% agarose gel in TBE buffer. Although the highmolecular weight E. coli genomic DNA and the E. coli RNA were digestedby the appropriate enzymes, there was no change to the signal of theArmored RNA™ except that it became more intense and resolved becausepresence of the E. coli nucleic acids in the supernatant tended to smearthe signal of the Armored RNA™. However, the nucleases did degrade thegenomic DNA and the host RNA.

Cells which were uninduced synthesized some Armored RNA™ but much lessthan the uninduced cells. Two cultures of pAR-1 in E. coli were grown tomid-log phase Cb₂₅-LB. One culture was then induced with 1 mM IPTG andthe other was not. Both cultures were grown for another 3 h, 37° C., 200rpm and then cultures were assayed for Armored RNA™ production. Asassayed by gel electrophoresis and Ethidium Bromide staining, moreArmored RNA™ was synthesized in the induced cells than in the uninduced.Cells were also induced for 16 h and this protocol resulted in theproduction of more Armored RNA™ than the 3 h induction. The ribonucleaseresistance of the Armored RNA™ containing the 1.7 kb truncatedbacteriophage RNA indicates that the full length 3.6 kb of the MS2bacteriophage genome is not required for the production of Armored RNA™particles.

2. Construction of an Armored RNA™ HIV Standard

pAR-1 served as the backbone for the creation of a quantitative HIV RNAstandard compatible with the Amplicor™ HIV Monitor™ kit (RocheDiagnostic Systems). This study shows one example of how Armored RNA™can be employed.

The QS RNA is the naked RNA standard in the Amplicor™ HIV Monitor™ test.The QS RNA encodes a conserved sequence of the gag gene from HIV butalso contains a 26 bp substitution of randomized sequence. This randomsequence is used to distinguish the wild type HIV amplicon from the QSamplicon since the HIV RNA and the QS RNA are co-amplified in theMonitor test.

RT-PCR™ was applied to the naked QS RNA to produce a DNA fragmentencoding the QS sequence and contained Bgl II and KpnI restrictionsites. The primers used were 5′ GATTGGTACCTGCTATGTCAGTTCCCCTTGGTTCTCT 3′(SEQ ID NO: 4) and 5′ GATTAGATCTAAGTTGGAGGACATCAAGCAGCCATGCAAAT 3′ (SEQID NO: 5). These primers correspond to the SK431 and SK462 primersrespectively used in the Monitor kit (Mulder, 1994) except that a Kpn Isequence was added to the SK431 primer and a Bgl II sequence wasincorporated into the SK462 primer. The QS PCR™ product was digestedwith Bgl II and Kpn I and ligated into the Bgl II and Kpn I sites inpAR-1 creating the recombinant plasmid, pAR-2 SEQ ID NO:6. DNA sequenceof the Nco I/Kpn I fragment of pAR-2.

An Nco I/Kpn I fragment containing the Maturase, the Coat Protein andthe QS RNA sequence was cloned into the Nco I/Kpn I restriction sites ofthe expression vector, pSE380 (Invitrogen Corp.) to produce pAR-2, SEQID NO:6. The important regions of the insert include: Nco I (1>6), BglII (1713>1718), Kpn I (1862>1867), Maturase coding sequence (53>1231),Coat Protein coding sequence (1258>1647), Lysis Protein (1601>1711), theCapture Sequence within the QS sequence (1757>1782), the QS Ampliconregion (1720>1861), SK462 primer (1720>1749), SK431 primer (1861>1835),Maturase Binding Site (311>337), and Operator sequence or pac site(1667>1687). SEQ ID NO:6.

pAR-2 in E. coli was induced with 1 mM IPTG in Cb₂₅-LB at mid-log phasefor 3 h, 37° C., 200 rpm. The induced cells were pelleted and thensonicated in the Sonication Buffer used for the AR-1 particles. Thencell debris was pelleted by centrifugation. The sonicate supernatant wasincubated with RNase 1 (5 units/μl) and DNase 1(0.1 units/μl), 37° C.,30 minutes and then fractionated on an agarose gel and visualized byEthidium Bromide staining. The AR-2 particles were detected as afluorescent band migrating at about 900 base pairs as compared to theDNA markers run on the same gel.

The AR-2 sonication supernatant containing the AR-2 particles wasincubated under the same conditions as above with RNase 1 and DNase 1except that the incubation was for 16 h. Another sample of AR-2 wasincubated 16 h, 37° C. but nucleases were not added. These samples werefractionated on an agarose gel and compared to the AR-2 preparationwhich was stored at 4° C. The AR-2 signal was strongest in the samplewhich was treated with the nucleases because the nucleases degraded thenucleic acids which were masking the AR-2 signal.

The AR-2 were also isolated from culture supernatant. pAR-2 transformedE. coli were induced with 1 mM IPTG, 16 h in 50 ml of Cb₅₀-LB medium.Inoculation and induction were simultaneous. 0.4 ml lysozyme (50 mg/ml)was added to the culture and incubated 37° C., 200 rpm, 1 h followed bythe incubation of the cell culture with 0.4 ml of1-bromo-3-chloro-propane 37° C., 200 rpm, 10 min. The cell debris waspelleted by centrifugation in an SS34 rotor, 4° C., 9000 rpm, 10 min.The culture supernatant was transferred to fresh tubes and 0.015 ml of1-bromo-3-chloro-propane was added. The culture supernatant was storedat 4° C. 0.015 ml of the culture supernatant was fractionated on anagarose gel and detected by ethidium bromide staining and UVfluorescence. AR-2 were detected in the supernatant as well as genomicDNA from E. coli. However, unlike the AR-2 isolated from E. colicytoplasm, this AR-2 preparation did not contain detectable E. coli RNA.A 0.5 ml prep of AR-2 from the spent growth medium was treated with 10units of DNase1, 37° C., 70 min. As assessed by gel electrophoresis,DNase1 completely degraded the host DNA but did not affect the AR-2.

0.02 ml of the AR-1 and AR-2 particles isolated from E. coli cytoplasmby sonication, were treated with 2 units DNase1 and 100 units RNase 1,37° C., 2 h and then 21° C., 16 h to degrade all host E. coli RNA andDNA. The AR preps were subjected to single tube RT-PCR™ using primerpairs specific for the QS amplicon, SK431 and SK462 (Mulder, 1994), andspecific for the MS2 bacteriophage sequence, MS2-1 and MS2-2. TheArmored RNA™ preps were diluted 10, 100 and 1000 fold in PBS. RT-PCR™was performed with 1 μl of each of the Armored RNA™ dilutions usingSK431/SK462 and MS2-1/MS2-2 primer pairs. Prior to the reversetranscription step, the Armored RNA™ preps were incubated at 95° C., 5min to disrupt the protective protein coat of the Armored RNA™. The RNAwas cooled on ice in the presence of the primers and then incubated with100 units MMLV-RT, 42° C., 1 h. 2.5 μl Taq polymerase was added,followed by PCR™. The PCR™ products were fractionated by gelelectrophoresis. Only the AR-2 generated the expected PCR™ product of142 bp with the SK primers whereas the AR-1 and AR-2 generated a 411 bpproduct using the MS2 primers. These results were consistent with theAR-2 particles containing the QS RNA target and an MS2 RNA target whilethe AR-1 only contain the MS2 sequence.

Of course, the above protocol can be modified to produce almost any RNAstandard by cloning the DNA sequence (encoding the RNA sequence ofinterest) into a recombinant plasmid such as pAR-1. SEQ ID NO:3. Thisplasmid encodes the Maturase, the Coat Protein and the Operator sequenceand there are many convenient restriction enzyme sites immediatelydownstream (3′) of the Bgl II restriction site of pAR-1. Theserestriction sites are the same restriction sites originally encoded inthe multiple cloning site of pSE380, from nucleotides 396 to 622. Thus,DNA fragments may be synthesized chemically for cloning into pSE380.Alternatively, PCR™ or RT-PCR™ may be used to synthesize long DNAfragments (>100 bp) using primers which introduce restriction sites atboth termini of the DNA fragment. The PCR™ DNA fragments may be digestedfor cloning into pAR-1.

An advantage of using the larger gene fragments is that PCR™ primers todifferent regions of these genes may be used with a single Armored RNA™standard and it is not necessary to construct a different Armored RNA™standard for each PCR™ primer pair that might be used. If largefragments of HIV RNA sequence (1 to 3 kb) are packaged into ArmoredRNA™, then the user has the option of using a variety of primer pairsfor performing RT-PCR™. This type of construct may more readily conformto the primer pairs that the researcher is currently using and he wouldnot need to change primer pairs in order to use the Armored RNA™standard. The user is not limited to just one set of primer pairs aswhen using, for example, the short 142 base pair region used in the HIVmonitor assay.

Of course, the HIV sequences may contain modifications from thewild-type sequence which will allow the standard sequence to bedistinguished from the wild-type for the purpose of using the ArmoredRNA™ in competitive PCR™ as quantitative standards. Such modificationsinclude insertions, deletions and restriction enzyme sequences.

It is not known what is the maximum limit of non-bacteriophage RNA canbe packaged as an Armored RNA™. However, the full length genome of MS2is ˜3.6 kb and pAR-1 encodes only ˜1.7 kb of the MS2 genome. Therefore,it is very likely that as a minimum, at least 2 kb of non-bacteriophageRNA sequence can be encapsidated as Armored RNA™ and possibly more ifthe Maturase and Coat Protein genes are supplied in trans.

3. AR-2 Used as a Quantitative RNA Standard for HIV

In the Amplicor™ HIV Monitor™ test, the naked QS RNA standard is addedto the Lysis buffer just prior to using the Lysis buffer for isolatingthe HIV RNA from plasma or serum. It is in this manner that the QS RNAis added to the plasma sample and that the HIV RNA and the QS RNA areco-purified. After isolation, the RNA is subjected to RT-PCR™ using TthDNA polymerase which functions as a reverse transcriptase and a heatstable DNA polymerase. After RT-PCR™, the PCR™ products are incubated inthe wells of an ELISA plate which contain oligonucleotides (captureprobes) immobilized to the well bottoms. One set of wells has anoligonucleotide sequence which is complementary to the wild-typesequence of HIV. The other capture probe recognizes the unique sequencein the QS amplicon. Thus, the HIV capture probe hybridizes to the HIVamplicon while the QS capture probe hybridizes to the QS amplicon whichresult from the RT-PCR™ co-amplification. After hybridization, theamount of amplicon hybridized is detected enzymatically andcalorimetrically using horse radish peroxidase. In the standard assayformat recommended by the manufacturers, about 60 to 80 copies of the QSRNA standard are added to each sample for analysis, depending on the kitlot number.

To test Armored RNA™ in the Amplicor™ HIV Monitor™ test, the naked QSRNA, and the concentrated AR-1 and the AR-2 were subjected to theAmplicor™ HIV Monitor™ test. The QS RNA, AR-1 and AR-2 were added to theLysis buffer, the RNA was isolated and then subjected to the RT-PCR™procedure. The PCR™ products were detected on assay plates. The QS RNAproduced the expected signal, the AR-1 were negative whereas the AR-2produced a signal so strong that it was off-scale. Once the concentratedAR-2 prep was diluted 30 million fold, the signal became comparable tothe QS RNA standard. The AR-2 prep was diluted by 30 million to producea stock of Armored RNA™ which was the equivalent of 8,000 copies of QSRNA per milliliter. 10 μl of this AR-2 stock produced the same signal inthe Amplicor™ HIV Monitor™ assay as the recommended amount of the nakedQS RNA standard provided with the kit.

The AR-2 were diluted in water and stored at 4° C. over a period ofseveral months. The diluted AR-2 were calibrated to produce a signalsimilar to the QS RNA in the Amplicor™ assay. Numerous Amplicor™ assayshave been performed with the same diluted stock of AR-2. During a 38 dayperiod, there has been no detectable decrease in the signal producedwith the AR-2, highlighting the durability of these RNA standards (FIG.1). This result is remarkable considering that this AR-2 stock was acrude preparation from E. coli and contained ribonucleases.

4. Durability of Armored RNA™ Compared to Naked RNA

One advantage of Armored RNA™ is that rather than adding the ArmoredRNA™ standard to the lysis solution prior to the lysis procedure, theArmored RNA™ standard may be added to the plasma or serum sample priorto RNA isolation, thus minimizing pipetting error in the assay. ArmoredRNA™ withstands plasma/serum nucleases very well compared to naked RNA.

10 μl RNA purified from AR-2 (about 5000 QS RNA equivalents), and intactAR-2 (about 500 QS RNA equivalents) were each added to 0.2 ml of normalhuman serum and incubated, 21° C. for 0 sec, 15 sec, 30 sec, I min, 5min, 15 min, 1 h, and 4 h. The incubations were stopped by the additionof 4M guanidine thiocyanate, and 1% Sarkosyl solution and the RNA wasextracted from the serum according to the method of Chomczynski (1987).Fifty microliters of a purified RNA preparation was amplified by RT-PCR™for 26 cycles using the SK431/SK462 primer pair. The PCR™ products werequantified with a solid phase ELISA system (Mulder, 1994).

The AR-2 generated the same signal over the full time course while thesignal from the purified RNA from the AR-2 disappeared almostimmediately (FIG. 2). Clearly, the RNA in AR-2 was protected againstplasma ribonucleases compared to the naked QS RNA.

EXAMPLE II Use of Armored RNA™ to Quantify HIV

1. Use of Armored RNA™ Standard in the Amplicor™ HIV Monitor™ Assay

To perform the Amplicor™ HIV Monitor™ assay, a quantitative RNA standardof a known quantity is added to the patient's plasma sample and then RNAis isolated from the plasma. RT-PCR™ is performed on the RNA such thatthe standard and the HIV RNA are co-amplified using a single primer set.Both PCR™ products are measured and then the concentration of the HIVRNA is calculated using the signal obtained from the quantitativestandard.

The Armored RNA™ standard containing the QS sequence (AR-2) is used inthe Amplicor™ HIV Monitor™ test as follows. A known quantity of AR2 (˜10μl) is added to 0.2 ml of sample plasma. 0.6 ml of Lysis Reagent isadded to the plasma sample containing the AR-2. The sample is mixed byvortexing 3-5 seconds and then incubated at room temperature 10 minutes.0.8 ml of isopropanol is added to the sample tube and the sample ismixed by vortexing 3 to 5 seconds. The sample is centrifuged at maximumspeed (˜16,000×g) for 15 minutes. The supernatant is discarded withoutdisrupting the pellet. 1 ml 70% ethanol is added, and the sample isvortexed 3 to 5 seconds followed by centrifugation for 5 minutes at˜16,000×g.

The pellet is resuspended in 0.4 ml of Specimen Diluent. 50 μl of theextracted sample is added to the MicroAmp tube containing the Master Mixand RT-PCR™ is performed using Tth Polymerase.

After amplification, 0.1 ml of Denaturation Solution is added to theamplicons. 0.1 ml of Hybridization Solution is placed into each of wellof the Microwell Plate (MWP) used for detection of the amplicons. 25 μlof the denatured amplicon is added to the first well and 1:5 dilutionsare performed using 25 μl in the next 4 consecutive wells such thatthere are dilutions of 1:5, 1:25, 1:125, 1:625 and 1:3125 for thedetection of the HIV amplicon. Dilutions of 1:5 and 1:25 are made in theappropriate wells for the detection of the QS amplicon. The MWP isincubated for 1 hour at 37° C.

The wells are washed 5 times with Working Wash Solution and then 0.1 mlof AV-HRP is added to each well and incubated 15 minutes, 37° C.

The MWP is washed 5 times with the Working Wash Solution. Then, 0.1 mlof the Working Substrate is added to each well and the MWP incubated 10minutes, in the dark, at room temperature. 0.1 ml of Stop Solution isadded to each well and the optical density is measured at 450 nm. Theconcentration of the HIV in the plasma is calculated based on the signalobtained from the known concentration of the QS standard added to thepatient plasma.

Besides using Armored RNA™ as a substitute for the naked QS RNA standardin the HIV Monitor™ assay, Armored RNA™ may also be used as a positivecontrol in the assay. A wild-type sequence of HIV compatible with theHIV Monitor™ test could be packaged in Armored RNA™ such that it behavesas if it were a wild-type HIV. In this embodiment, an Armored RNA™ HIVPositive control is added to normal plasma at a known quantity and isthen processed as if it were a patient sample except that the user wouldexpect to obtain a certain pre-determined value in the assay. Thisstandard would be used to demonstrate to the user that the assay wasfunctioning properly.

2. Modified Amplicor™ HIV Monitor™ Test Procedure Well Suited to ArmoredRNA™

To increase the sensitivity of detection for HIV in the Amplicor™ assay,a procedure was developed in which the HIV virions are pelleted by highspeed centrifugation from the plasma sample. Thus, 1 ml of plasma cannow be assayed instead of the conventional 0.2 ml which the assay hasused. This procedure should theoretically increase the sensitivity 5fold, if the centrifugation step pellets 100% of the HIV virion. Thisprotocol is only useful as a qualitative assay for HIV.

In this new procedure, the naked QS RNA would not be an optimalstandard. It is cannot be added to a plasma sample due the ribonucleasesin the plasma which would degrade it and the naked QS RNA would notpellet by centrifugation with the HIV virions. Thus, when the plasma isremoved after centrifugation, there is no control for loss of the HIVpellet. Armored RNA™ may be added to the plasma prior to centrifugation.It should pellet similarly to the HIV. Thus any loss of the pellet wouldbe reflected in an equal loss in signal obtained from the Armored RNA™standard. Importantly, the use of Armored RNA™ standards can convert thecentrifugation protocol from a qualitative assay to a quantitativeassay.

3. Use of Armored RNA™ in Other Assays

Of course, the invention is not limited to a single, exemplary assay.There are other RNA based assays for HIV including: NASBA which is basedon isothermal amplification (van Gemen, 1994; sold by Organon Teknika);the branched DNA assay developed by Chiron; an assay by DiGene in whichan antibody recognizes a DNA/RNA duplex; and transcription mediatedamplification, a technology similar to NASBA developed by Gen-Probe. Foreach of these assays, one skilled in the art can construct an ArmoredRNA™ standard containing appropriate sequence(s) to functionappropriately as a quantitative standard.

The HIV NASBA assay uses three different quantitative RNA standards. Thesequences of these standards are available in van Gemen (1994). Each ofthese NASBA standards could be cloned into pAR-1 in the same manner thatthe QS sequence was cloned into pAR-1 to produce Armored RNA™ standardsfor the NASBA assay. RT-PCR™ may be used to amplify each of thestandards for cloning into pAR-1.

The branched DNA assay by Chiron uses a single stranded DNA as the RNAstandard. It encodes the gag and pol genes of HIV strain SF2. Thesequence for this strain of HIV is available from GenBank, Accessionnumber K02007. This HIV standard sequence is ˜3 kb and may be too longfor its full length to be packaged using pAR-1 as the vector for ArmoredRNA™ synthesis. It may be necessary to use a different is constructwhich may permit longer sequences to be packaged. One such Armored RNA™composition would be comprised of Maturase protein, Coat Protein and areRNA coding Coat Protein, Operator site(s) Maturase Binding Sites andthe HIV sequence encoding gag and pol. The Maturase would be encoded onanother plasmid provided in trans. In this construct, the deletion ofthe Maturase Coding sequence from the reRNA 20 may permit for thepackaging of more non-bacteriophage sequence.

EXAMPLE III Use of Armored RNA™ in a HIV Gel-Based Assay

An Armored RNA™ may be used as an external quantitative standard. Afragment of the HIV genome such as that region bounded by the SK431 andSK462 primers or some other region such as sequence encoding the polgene can be cloned into the pAR-1 backbone. This construct can be usedto synthesize Armored RNA™ in which the entire length of the cloned HIVfragment is wild-type HIV sequence. Such an assay does not require aunique capture sequence, because each amplicon is detected on anacrylamide or agarose gel either by ethidium bromide staining or bylabeling the PCR™ product with ³²P incorporation and autoradiography. Astandard curve is generated by introducing known amounts of the HIVsequence in to the RT-PCR™ assay and then quantifying the amount ofproduct generated on a gel. Actual copy numbers for test samples maythen be derived using the standard curve. The Armored RNA™ is calibratedand used in RT-PCR™ reactions as if it were actual HIV. The PCR™fragments may be fractionated either by agarose or acrylamide gelelectrophoresis and quantified by ethidium staining or radioactivity.Standard curves may be generated by processing plasma containingdifferent concentrations of the Armored RNA™. The standard curve maythen be used to calculate the titers of patient samples byinterpolation.

EXAMPLE IV Use of Armored RNA™ as a Non-Infectious Standard

Armored RNA™ may be used as non-infectious proficiency standard (acertified and well characterized plasma, serum or urine based productdesigned for the validation is of the accuracy of the instrumentationand methods of an assay) or as positive controls. Gene fragments of astandard HIV strain are cloned into the Armored RNA™ recombinant plasmidto produce a family of Armored RNA™-HIV standards. These include the poland gag genes, which are well conserved in HIV. The inventors envisionAR-gag, AR-pol and AR-gag/pol RNA standards. These Armored RNA™standards are quantified precisely and then can be used as externalquantitative standards for RT-PCR™ or other amplification techniques.The Armored RNA™ may be added directly to plasma and used as such, orthe AR-HIV RNA may be extracted from the purified Armored RNA™ in astandard salt buffer such as PBS or Tris:NaCl.

EXAMPLE V An Armored RNA™ Standard Used in an HCV Assay

Armored RNA™ technology will be useful in creating RNA standards forviruses that are difficult or hazardous to culture. For example,hepatitis C virus (HCV) cannot be reliably grown in tissue culture,whereas an Armored RNA™ standard for HCV could easily be constructed andproduced. HCV isolates can be classified into 6 distinct genotypes basedon the nucleotide sequence variation in the core gene region or the NS5region (Simmonds, 1994). Genotype-specific Armored RNA™ standards can beconstructed to serve as controls in assays designed to identify specificHCV genotypes and HCV subtypes such as 1a, 1b, 2a, 2b, 2c, 3, 4a, 4b,5a, and 6a. Genotyping strategies have been based on sequencing, RFLPanalysis, PCR™-based assays with type-specific primers, and a line probeassay (van Doom, 1994). For example, the latter assay involves thedifferential capture of a 244 basepair product that is generated fromprimers at positions 56 through 299 of the HCV genome. The amplicon usedto identify each of these strains is short enough that it isstraightforward to chemically synthesize the DNA fragments to be clonedinto pAR-1 and thus circumvent the need to handle infectious material inorder to clone these genotype sequences. Protected recombinant RNA fromhuman serum samples could be constructed using primers KY80(5′GCAGAAAGCGTCTAGCCATGGCGT) (KY80-SEQ ID NO: 7) and KY78(5°CTCGCAAGCACCCTATCAGGCAGT) (KY78-SEQ ID NO: 8). By using samples withdefined genotypes, genotype-specific recombinant standards could beconstructed. Armored RNA™ standards for HCV may be constructed using astrategy similar to the one for the HIV standard. These RNA standardsmay be used either as positive controls for strain typing orquantitative RNA standards.

For strain typing, the primers KY78 and KY80 may be used to synthesizeDNA fragments by RT-PCR™ from different HCV strains. These strainspecific DNA fragments may be cloned into pAR-1 such that RNA encodingstrain specific HCV is packaged as Armored RNA™. As well, strainspecific sequences have also been documented for HCV between positions56 to 299 of the HCV genome. It is possible to chemically synthesizethese sequences and clone them directly into pAR-1 for packaging. Thismethod would circumvent the need to handle infectious HCV. Thus, in thedifferential capture assay, each strain specific PCR™ product of HCVwould hybridize to a unique capture probe immobilized to the bottom of aplastic well.

To create a quantitative HCV Armored RNA™ standard, the ampliconproduced by the KY78 and KY80 primers could be modified by substitutingin the same capture sequence used in the QS sequence for HIV or someother 25 to 30 base pair region within this amplicon may be randomizedso that it can be differentiated from the wild-type amplicon. Thesubstitution may be performed by one skilled in the art by PCR™. As inthe Amplicor™ HIV assay, the HCV quantitative standard would beco-amplified with the wild-type RNA. The HCV Armored RNA™ standard wouldbe added to the sample plasma prior to the RNA isolation step.

EXAMPLE VI Collection Tubes Containing Pre-Determined Quantity ofArmored RNA™

Owing to the durability and stability of Armored RNA™, it may bealiquoted into blood collection vessels or other fluid collectionvessels in a pre-determined quantity. The Armored RNA™ may then befreeze dried for long term storage at room temperature or left inbuffered salt solution and stored at 4° C. or even room temperature(˜21° C.). At Is the time of use, blood is drawn into the collectiontube with the Armored RNA™ standard. The blood sample is inverted manytimes to thoroughly mix the Armored RNA™ standard into the sample. Theblood may be stored as usual until it is subjected to the quantitativeassay. This strategy precludes the need to add an RNA standard to theplasma sample prior to the RNA isolation from plasma. It would controlfor the partitioning of the viruses between the blood cells and theplasma or serum fractions.

Further, multiple Armored RNA™ standards may be included in thecollection tube such as an HIV and an HCV standard so that thequantification for one or both pathogens may be performed.

EXAMPLE VII Use of Armored RNA™ in Veterinary Diagnostics

Domestic animals are often infected with RNA viruses. For cats,retroviruses represent the largest cause of premature death other thanautomobile accidents. Up to one-third of the cats exposed, are infectedwith Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus(FeLV) (Essex, 1995), similar to their human counterparts, HIV and humanT cell lymphotropic virus-type 1 (HTLV). One skilled in the art couldconstruct Armored RNA™ standards for FIV and FTLV, similar to the AR-2standard described for HIV. These standards may be used forquantification or as positive controls in FIV and FeLV diagnosis byRT-PCR™.

EXAMPLE VIII Use of Double Stranded RNA Bacteriophage

Bacteriophage φ6 is a double stranded RNA bacteriophage in which it ispossible to package in vitro, non-bacteriophage RNA to produce viable,genetically stable bacteriophage (Qiao, 1995). It has been demonstratedthat the RNA in these bacteriophage are protected from ribonucleases.One skilled in the art may produce recombinants containing a standardRNA sequence so that the recombinant bacteriophage φ6 may act asquantitative standards for human infectious, double stranded RNA virusesis such Rota Virus and Vesicular Stomatitis Virus. It is possiblefollowing the teachings of this specification and the art to develop apackaging system with bacteriophage φ6 whereby the packaged material isnot viable/infectious, similar to the Armored RNA™ standards.

A double stranded non-infectious RNA bacteriophage may have applicationsas a therapeutic. Double stranded RNA is known to stimulate cells toproduce important immunomodulators which act as anti-viral agents suchas interferon (Stiehm, 1982).

EXAMPLE IX Quantification of a Cellular mRNA

One skilled in the art can use an Armored RNA™ standard for thequantification of an mRNA expressed by a cell. In studies to determinethe induction or repression of specific mRNAs over a period of time, anequal number of cells could be harvested at each time point before andafter the cells had been exposed to the treatment under investigation.The Armored RNA™ standard would be added in a known quantity to eachtime point prior to the purification of the RNA from the cells. TheArmored RNA™ standard could then be used to quantify the amount of atarget mRNA such as a cytokine, a cell cycle gene or an oncogene. TheArmored RNA™ standard would be constructed containing the same primerpair binding sites as the target gene. The Armored RNA™ standard couldbe differentiated from the wild type RT-PCR™ product by altering thesequence between the primers pairs using one of the methods discussedabove such as incorporating a restriction site or a deletion. Oneskilled in the art can produce amplicons which have insertions,deletions or substitutions as compared to the wild-type sequence. Thereare several different methods available for site-directed mutagenesis,each of which involve oligonucleotides which encode the desired sequenceof the standard.

Standards containing deletions, insertions and restriction sites areoften preferred for gel based assays because they are easilydifferentiated from the wild-type amplicon by fractionation on anagarose or acrylamide gel. For each of these standards, a sizedifference of about 10% is often used. PCR™ may be used to generate anyof these standard sequences. To generate a deletion mutant, anoligonucleotide is synthesized which contains primer sequences as wellas other amplicon sequence. The amplicon sequence and the primersequence flank the sequence in the wild-type sequence which is to bedeleted. When this oligonucleotide hybridizes to the wild-type, thesequence to be deleted is looped out and is not incorporated into thenew DNA strand during polymerase extension. One skilled in the art mayuse similar strategy to create insertions and substitutions whereby oneof the primers in a PCR™ reaction contains the desired mutation. Twosuch strategies are discussed in detail in Schneeberger (1996) andHughes (1996).

EXAMPLE X Preparation of Armored RNA™ Standards for Commercial Use

It is important that the Armored RNA™ standard is free of any host DNAor RNA which may affect quantification by RT-PCR™. To produce ahomogenous lot of an Armored RNA™ standard, a crude extract would beprepared from the culture supernatant after a 16 h induction of the E.coli transformants (see EXAMPLE I). This preparation is contaminatedwith the genomic DNA from E. coli. Contaminating RNA and DNA may beremoved by adding RNase and DNase to the crude extract.

Traditionally, MS2 bacteriophage are purified by a CsCl gradient. Theyband tightly at a concentration of 1.45 g/cc (Pickett, 1993). ArmoredRNA™ may be purified by using a CsCl gradient procedure similar to theMS2 bacteriophage. After centrifugation, the Armored RNA™ band is pulledand then dialyzed against a salt buffer such 100 mM NaCl: 50 mM Tris (pH7.5) or PBS. Armored RNA™ may be quantified by obtaining an OD₂₆₀ and anOD₂₈₀ which are used in the art to measure nucleic acid and proteinconcentrations, respectively. After a stock of Armored RNA™ has beenmade, it may be calibrated against a naked RNA containing the sameamplicon. For example, Armored RNA™ standards may be calibrated againstthe naked QS RNA standard.

The nuclease treated crude extract of Armored RNA™ may be also purifiedby gel exclusion chromatography, using a resin such as Sephacryl S-200(Pharmacia) (Heisenberg, 1966). Due to the large size of the ArmoredRNA™, they will run in the void volume while other protein and nucleicacid components will be retarded. Armored RNA™ in the void volume may becalibrated as above. It will be advantageous to couple severalpurification procedures to ensure that the Armored RNA™ is completelyhomogenous.

Large scale production of Armored RNA™ may be performed as follows. A100 ml inoculum of E. coli harboring the pAR construct is grown tomid-log phase in Cb₂₅-LB medium. This inoculum is used to inoculate1,000 ml of 1 mM IPTG-Cb₂₅-LB medium. The cells are incubated 16 h, 200rpm, 37° C. One ml of lysozyme (50 mg per ml) is added to the cultureand incubated 37° C., 1 h, 200 rpm. The culture is pelleted bycentrifugation. The supernatant contains Armored RNA™ particles and E.coli genomic and plasmid DNA. Contaminating RNA and DNA is degraded byadding CaCl₂ to the supernatant to 10 mM and the supernatant is thenincubated with Micrococcal Nuclease (30 units/ml), 37° C., 1 h, 200 rpm.EDTA is added to 25 mM to chelate the CaCl₂ and stop the nucleaseactivity. The AR particles are precipitated with 50% Ammonium Sulfate,4° C., 2 h. The precipitate is pelleted by centrifugation. The pellet isresuspended in 100 mM NaCl: 1 mM EDTA: 10 mM Tris (pH 7.5) (TSE buffer).0.6 g of CsCl is added to every gram of AR2 solution. The CsCl isdissolved and transferred to a heat sealed ultracentrifugation tube forthe 50.2 Ti rotor (Beckmann). The CsCl gradient is centrifuged at 45,000rpm, 20 h, 21° C. The AR particles band about the middle of thecentrifuge tube. The CsCl band is pulled with a needle and syringe,about 5 ml. The AR2 particles are finally passed over an Sephacryl S-200resin in TSE buffer. The particles elute in the void volume. The numberof AR2 particles may be determined using the extinction coefficient of 1OD₂₆₀=0.125 mg/ml of MS2 bacteriophage and the molecular weight is3×10⁶. Based on this procedure, approximately 1×10¹⁵ AR particles can bepurified from one liter of culture.

Electron microscopy may be used to count the Armored RNA™ directly. Thismethod has been used for quantifying HIV (Lu, 1993).

After purification, the Armored RNA™ may be stored at 4° C. or at roomtemperature. A biocide may be added to prevent bacterial or fungalgrowth in the standards. The Armored RNA™ is diluted to concentrationswhich are convenient for quantification.

EXAMPLE XI Use of other RNA bacteriophage for constructing Armored RNA™

There are 3 other genetically distinct groups of RNA coliphage and atleast 2 other non-E. coli RNA bacteriophage, PP7 and PRR1 (Dhaese, 1979;Dhaese, 1980). These bacteriophage have Coat Proteins which arehomologous in protein sequence to the MS2 Coat Protein (Golmohammadi,1993). As with the MS2 phage, the cDNA of the Maturase and the CoatProtein of any of these bacteriophage could be cloned into an expressionvector such as pSE380. These recombinant plasmids may act as vectorsinto which non-phage DNA sequence may be cloned. The transcript of thenon-phage sequence would contain the Operator sequence and the Maturasebinding sequence for these other bacteriophages such that the non-phageRNA sequence would be encapsidated with Coat Protein and Maturase of theappropriate strain specific phage. In this manner. Armored RNA™ may becomposed of Coat Protein and Maturase from any of the RNA bacteriophagespecies. Similarly to the pAR-2 construct, induction would produce areRNA which encodes Maturase, Coat Protein, Operator sequence(s), andthe Maturase Binding sequence(s) of these other bacteriophages such thatthe non-bacteriophage RNA sequence would be encapsidated with the CoatProtein and Maturase of the appropriate strain specific phage.

EXAMPLE XII

Plant Virus Armored RNA™

Plant viruses can be used to prepare Armored RNA™. For example, TMV andPotyvirus can be used for this purpose.

1. Tobacco Mosaic Virus Packaging.

It is possible to package recombinant RNA (reRNA) using Tobacco MosaicVirus (TMV) coat protein in E. coli (Hwang 1994a and Hwang 1994b). TMVis a filamentous virus composed of 2,100 of a 17 kDa coat protein thatprotects a 6.4 kb single stranded, positive sense genomic RNA againstribonucleases. TMV has been used as a model system for studyingself-assembly of multimeric biological structures. Under the correctconditions, purified TMV coat protein and the gRNA will spontaneouslyassemble to form an infectious virus in vitro. As well, reRNA packagedin vitro with TMV coat protein is protected against ribonucleasedigestion (Jupin et al., 1989).

The TMV gRNA has an operator-like sequence called theOrigin-of-Assembly-Sequence (OAS) recognized by the coat protein toinitiate assembly. The OAS is located at nucleotides 5112-5543 in thewild-type virus, 432 nucleotides in length. The OAS exists as a threestem-loop structure. The minimal length of the OAS required forpackaging is 75 nucleotides, nucleotides 5444-5518. The coat proteinassembles faster in the 3′ to 5′ direction along the RNA than in the 5′to 3′ direction.

In the TMV-E. coli packaging system, TMV coat protein was plasmidencoded and provided in trans to the reRNA to be packaged. (Hwang 1994a,Hwang 1994b, U.S. Pat. No. 5,443,969). reRNAs, containing a 5′open-reading frame and an OAS, of 1.6 kb (CAT-OAS RNA) and 2.8 kb(GUS-OAS RNA) were transcribed in E. coli from a second plasmid.Co-expression of the coat protein and the reRNA resulted in thepackaging of the reRNA. The integrity of the reRNA packaged was assayedby RT-PCRT™. Full-length CAT-OAS RNA was detected by RT-PCR™ butfull-length GUS-OAS RNA was not detected.

2. Potyvirus Packaging.

Potyvirus is another example of a plant virus that can be used toproduce Armored RNA™. Its coat protein has been expressed and assembledinto capsids in E. coli (Zhoa, 1995). However, there was no mention ofthe RNA which may have been packaged. Presumably, there must be anoperator-like sequence as is found for MS2 and TMV. No reference hasbeen found to such-a sequence for Potyvirus. A reRNA could be packagedby this coat protein once its operator sequence was discovered.

To define the operator sequence in potyvirus, a systematic set ofpackaging experiments may be performed, similar to the experiments ofPickett and Peabody (1993). The potyvirus coat protein gene may beexpressed from one vector in E. coli. Another vector may be used totranscribe different segments of the potyvirus genomic RNA in about 1 kblengths. These sequences could be fused to a reporter sequence, that is,a known sequence common fused to each segment of the potyvirus genomicRNA. The reporter sequence may be as short as 20 bases. The potyviruscoat protein and the test sequences may be co-expressed in E. coli andthe viral particles isolated. The RNA may be isolated from the particlesand then the RNA assayed for the reporter sequence by one of a number ofdifferent methods such as RT-PCR™, Northern blotting, dot blotting, orRPA. Particles containing the reporter sequence would be candidates forcontaining the operator sequence. The operator sequence can be furtherdefined by performing an iteration of the same experiment, furtherdividing the 1 kb candidate sequence into 250 base sequences. Once anoperator sequence is identified, it can be used with potyvirus coatprotein to package heterologous RNA sequences.

By employing a strategy like that used to make bacteriophage ArmoredRNA™, a recombinant RNA can be transcribed in E. coli composed of thepotyvirus coat protein sequence, non-potyvirus sequence and thepotyvirus operator sequence. This reRNA will be packaged specifically bythe potyvirus coat protein because it contains the operator sequence atthe 3′ end. In this system, the coat protein sequence and the operatorsequence are in cis. Another alternative is to transcribe the coatprotein RNA and the non-potyvirus RNA/operator RNA in trans. Such atrans system was used both with MS2 (Pickett and Peabody, 1993) and withTMV.

EXAMPLE XIII Animal Virus Armored RNA™

Animal viruses can be used to produce Armored RNA™. An example of thisis seen with regard to the alphavirus.

1. Alphavirus Packaging.

Alphaviruses are enveloped positive, single strand RNA viruses whichinfect is mammals, birds, and insects. Examples from this virus familyare Semliki Forest Virus (SFV), Sindbis virus, and Venezuelan EquineEncephalitis (VEE). Over the past ten years, these viruses have beenadapted for the expression of recombinant RNAs and proteins in animalcells. (Frolov 1996 and U.S. Pat. No. 5,217,879)

The alphavirus particle has a single genomic RNA protected by 240 copiesof basic capsid protein (C), surrounded by a lipid bilayer containing240 E1E2 envelope glycoprotein heterodimers. Alphaviruses can infect avariety of cell types and may use more than one receptor to gain entryinto a cell. The virus has an icosehedral symmetry. The genomic RNA iscapped at the 5′ end and it is organized such that the 5′ end halfencodes non-structural genes for RNA replication and the 3′ half encodesthe structural genes for packaging the RNA. There is a packaging signalsequence within the non-structural region of the genomic RNA requiredfor efficient encapsidation.

Several strategies have been used to package reRNA using the alphavirussystem. In one method, an infectious recombinant virus is produced inwhich the complete wild-type genomic RNA is maintained and the foreignsequence of interest is inserted 5′ or 3′ or the structural genes.Foreign RNA sequences up to 2 kb can be packaged. This method was usedto package several foreign genes including bacterial chloramphenicolacetyltransferase (CAT) and a truncated form of the influenzahemaglutinin. Cells infected with these recombinant alphavirusesproduced the encoded proteins.

Another strategy for the packaging of reRNA involves the production ofnon-infectious particles. In this system, the structural andnon-structural RNAs are encoded in separate, distinct RNAs with thereplicase sequence containing the packaging signal and the foreign RNAsequence. These two RNAs are co-transfected-into a cell. The replicasereplicates both the non-structural and structural RNAs while thestructural RNA generates the proteins which only package thenon-structural/foreign gene RNA. These particles are non-infectiousbecause the structural genes are not packaged with the non-structuralgenes. Using this method, it is estimated that at least 5 kb of foreignRNA can be packaged.

There are other variations of the above described packaging systemsinvolving similar type strategies but most of them are designed toincrease the protein expression of the foreign RNA sequence.

The alpha-particles will protect RNA from ribonucleases in much the samemanner as the MS2 particles. It is to be expected that the reRNA willprotected from ribonucleases because alphaviruses could not re-infect iftheir genomic RNA were susceptible to ribonucleases once it was packagedand the viruses were released from their host cell.

The alpha-particles may not be as heat resistant as bacteriophageArmored RNA™, because they have a different coat structure. Thebacteriophage Armored RNAs may be the preferred choice for shippingpurposes if alphaviruses are heat sensitive. However, any heatsensitivity would not be an insurmountable problem as particles couldsimply be shipped on dry or wet ice.

Because alphaviruses are physically very similar to HIV, they may be abetter pelleting standard for ultra-sensitive assays. The alphaviruseshave more in common with HIV and HCV structurally than with thebacteriophages. Thus, they may pellet by centrifugation more like theHIV and HCV they are supposed to mimic. Pelleting the viruses may beimportant in developing a good standard for an ultrasensitive assay. HIVin 1 ml of plasma can be concentrated by centrifugation and then the RNAis extracted from the viral pellet. Alphavirus particles may also acceptlonger foreign sequences than the MS2 system, because they have a largergenome than bacteriophage. Alphavirus particles should be stable inserum/plasma for time periods similar to HIV. Many of these viruses aretransmitted through mosquito vectors. Therefore, the virus must surviveexposure to plasma.

EXAMPLE XIV Construction of Armored DNA

There are many well studied single- and double-stranded DNAbacteriophage which infect E. coli as well as a number of otherbacteria. It is straightforward to clone DNA fragments into a singlestranded bacteriophage such as M13 or into the double strandedbacteriophage λ. These recombinant phage are readily purified andquantified by determining the plaque forming units. They may act asstandards for PCR™ based assays for DNA viruses. For instance, a λrecombinant bacteriophage containing a conserved DNA sequence of thehuman pathogen, the Herpes Simplex Virus may act as a double strandedDNA standard. It may be used as a quantitative standard or as a positivecontrol.

EXAMPLE XV Subtraction of rRNA from total RNA

The Armored RNA™ may be used to synthesize large mass amounts of RNAwhich encodes the antisense sequence of 18S and 28S rRNA. Once the RNAhas been isolated and purified, it can be labeled with photobiotin. Thebiotinylated RNA is added to total RNA in molar excess over the target18S and 28S rRNA. The mix is heat denatured in a hybridization bufferpromoting the formation of RNA hybrids and then allowed to cool to roomtemperature to encourage the formation of the duplex between the 18S and28S rRNA with the biotinylated antisense RNA. After hybridization,streptavidin is added to the solution and binds to the biotinylated RNA.A phenol extraction partitions all the unbound streptavidin andbiotin/streptavidin complexes into the organic phase. The 18S and 28SrRNA hybridized to the antisense biotinylated RNA also partitions intothe organic phase. By this strategy, the 18S and 28S are subtracted fromthe total RNA. Since the molar quantity of 18S and 28S rRNA is veryabundant in total, large quantities of antisense RNA would be requiredfor this strategy. Armored RNA™ technology could provide as a means toprovide a cheap source of RNA.

EXAMPLE XVI Construction of RNA Size Standards

Mixtures of RNA of very discrete lengths are often used as RNA sizestandards for gel electrophoresis. These size standards are used oftenin Northern blotting to estimate the length of unknown mRNA species. Ingeneral, the RNA size standards range from 0.2 to 10 kilobases. The RNAin these standards are susceptible to ribonucleases and they are usuallyproduced by in vitro transcription. Therefore, any of the methodsdescribed herein may be used to create ribonuclease resistant RNA sizestandards.

For example, a series of Armored RNA™ standards can be constructed, eachcontaining an RNA of a different length. DNA fragments of differentsizes would be cloned into pAR-1 to construct this series of sizestandards. The size range of the standards will be dependent on the RNAsize limitation of the packaging system. After production, each of theArmored RNA™ size standards would be produced and quantified separately.The standards would be mixed so that the RNA of each of the standardsare at the same mass concentration.

In a preferred embodiment, the mixed Armored RNA™ size standards will beadded to a denaturing solution just prior to their use for gelelectrophoresis in order to disrupt the RNA-protein complex. Thedenaturing solution may consist of an acid such as acetic acid, adetergent such as SDS or a chaotrope such as urea. The standards wouldbe heated and then loaded on the gel. The advantage of this embodimentis that there is no chance for the RNA standards to be degraded untiljust prior to gel electrophoresis.

In another embodiment, the Armored RNA™ mixture is used as the stocksolution from which to isolate the size standards. The RNA may beisolated using a common RNA isolation procedure such as by Chomczynski(1987).

Of course, it is also possible to isolate the RNA from each of theArmored RNA™ size standards separately and then mix the RNA afterwards.

Alternatively, it is possible to construct ribonuclease resistantchemically modified RNA size standards, which can be used as a singlestandard or in sets of mixed standards.

EXAMPLE XVII Removal of the Non-Phage RNA from the Phage RNA

There are applications where a researcher may want an RNA which does notencode any phage sequence. All the Armored RNA™ compositions contain areRNA which encodes some amount MS2 bacteriophage sequence. One skilledin the art may construct the RNA sequence so that the RNA sequence ofinterest is flanked by ribozyme sequences as demonstrated previously(Ferré-D'Amaré, 1996). The non-phage RNA may be encapsidated with theribozyme sequences because these sequences are not very active in E.coli. After the RNA is isolated from the Armored RNA™, the RNA may besubjected to buffer conditions which produce optimal ribozyme activity.After ribozyme cleavage, the RNA fragment of interest may be purified bygel electrophoresis or HPLC.

Another method previously used by others involves using Ribonuclease Hwhich cleaves RNA at RNA/DNA duplexes (Lapham, 1996). Oligonucleotidescomplementary to regions flanking the desired RNA fragment arehybridized to the naked reRNA after it has been purified from theArmored RNA™ particle. This substrate is subjected to RNase H which willcleave the reRNA at the sites where the oligonucleotide has hybridized.DNaseI may be used after to digest all the oligonucleotides.

After purification, the RNA fragment may be capped with RNAguanyltransferase using GTP and SAM which may then be a suitablesubstrate for translation.

EXAMPLE XVIII Use of Armored RNA™ to Deliver RNA In Vitro and In Vivo

Armored RNA™ technology may be used to deliver intact, full length RNAto cells in vitro and in vivo. Transfection of cells with RNA isperformed with naked RNA which is susceptible to ribonucleases.Transient gene expression may be optimized by producing an RNA messageas an Armored RNA™, to protect the RNA until it enters a cell. Uponentry in the cell, the protective Coat Protein may be removed by thecellular machinery. The use of a bacteriophage system to deliver intactRNA would be similar to a TMV system which was previously employed. Arecombinant RNA was packaged in vitro using TMV Coat Protein andsuccessfully delivered into plant cells and into frog cells (Gallie etal, 1987).

The RNA need not be capped for translation. An encephalomyocarditisvirus or Polio translational leader sequence may be fused upstream ofthe coding sequence to promote the internal initiation of translation invitro or in vivo (Elroy-Stein, 1989). This system is cap independent fortranslation. The Armored RNA™ may be mixed with DOTAP, Lipofectin,Transfectam or Lipofectamine to form cationic liposomes to optimizemembrane fusion. Liposomes have been used successfully to increase theefficiency of RNA transfection with cells from tissue culture (Lu, 1994;Dwarki, 1993). As well, the Maturase or the Coat Protein could begenetically engineered so that either or both contain a peptide sequencewhich recognize specific cell surface receptors. These ligands wouldpromote tissue-specific uptake of the Armored RNA™. Peptide sequences upto 24 amino acids can be inserted into the MS2 Coat Protein such thatthe recombinant Coat Protein will maintain its ability to assemble intocapsids (Mastico et al., 1993). Peptide sequences which recognizecell-specific receptors could be incorporated into Coat Protein. Therecombinant Coat Proteins would be used to package reRNA, in vitro or invivo, into Armored RNA to produce a multivalent macromolecule whichwould recognize specific cell surface receptors. The multivalentproperty of the Armored RNA would confer a strong affinity for the cellsurface receptor. The peptide receptor sequences could be derived usingphage display technology (Barry, 1996; Pasqualini, 1996) or by usingpreviously characterized peptide sequences (Hart, 1994).

An mRNA may be packaged in vivo by cloning the gene of interest, such asthe CEA gene (associated with many different tumors and thought to be atumor rejection antigen), into a vector such as pAR-1. The gene would becloned immediately downstream of the Operator sequence in pAR-1. Aswell, the EMCV translation sequence would be cloned between the Operatorand the CEA coding sequence. In this way, the CEA RNA may be translatedwithout requiring a 5′ CAP to enhance translation of the mRNA.

For in vitro transfection, the CEA-Armored RNA™ would be mixed with thetissue cultured cells, with or without forming liposomes beforetransfection. The liposomes may enhance fusion of the Armored RNA™ withthe cells. Also, the CEA-Armored RNA™ may be microinjected into oocytes.The use of Armored RNA™ would forgo the requirement for the usualprecautions in handling RNA. In these procedures, it is expected thatthe Armored RNA™ would dissociate upon entering the target cell andrelease their packaged RNA so that it is available for translation.

In vivo, the Armored RNA™ may be injected into tissues by a variety ofroutes such as intravenous or intramuscular to produce the protectiveimmune response. It would be desirable that the Armored RNA™ could betaken up by the appropriate immune cells to produce the strongest andmost protective immune response. One skilled in the art could introducethe appropriate counter receptor (or ligand) sequence into the Maturaseor Coat Proteins so that the Armored RNA™ would bind and be taken up bythe strongest immunomodulators. This strategy would require priorknowledge about the some specific cell specific receptors on theseimmunomodulators so that they could be targeted appropriately.

If a one time vaccination is not enough to produce a protective immuneresponse, then multiple immunizations may be required. In this case, theArmored RNA™ themselves may be immunogenic and would become lesseffective at transfection as the individual develops immunity to theArmored RNA™ itself. Multiple vaccinations may involve using ArmoredRNA™ developed from RNA bacteriophage from several different serotypes.All of the Armored RNA™ vaccinations would contain the same protectivemRNA, however, it would be packaged by phage capsids from differentserotypes. For instance, the RNA coliphages are divided into serologicalgroups I, II, III and IV. There are also the two strains of P.aeruginosa RNA bacteriophase. Thus, the CEA mRNA may be packaged in asmany as 6 different Armored RNA™ and each would be used for eachdifferent immunization.

A capped mRNA may be encapsidated co- or post-transcriptionally. An mRNAwith a 5′ cap may be synthesized by using a cap analog nucleotide duringin vitro transcription or an RNA transcript may be capped with RNAguanyltransferase using GTP and S-adenosyl methionine as substrates.After mRNA synthesis, the mRNA may be mixed with Coat Protein and/orMaturase to produce Armored RNA™. The mRNA may contain one or moreOperator sequences and/or one or more Maturase Binding sites but theyare not essential for the packaging of long RNA molecules. These ArmoredRNA™ synthesized completely by in vitro methodologies may be used fortransfection into cells. Again, the Coat Protein may manipulated toexpress a ligand which will enhance the binding of the Armored RNA™ tothe target cell and promotes its uptake and processing.

Recently, phage display technology was used to select for peptides whichbound selectively to specific cell types in vitro (Barry, 1996) and invivo (Pasqualini, 1996). Libraries of filamentous DNA bacteriophagedisplaying random peptide sequences were either panned over mousefibroblast cells grown in culture or they were injected intravenouslyinto mice and the bound bacteriophage were eluted from different organs.Panning over the mouse fibroblast cells resulted in bacteriophage cloneswhich bound well to fibroblast, hepatoma, and mastocytoma cells but notto myoblast or macrophage cells. Intravenous injection into a mouseresulted in bacteriophage which bound selectively to brain tissue and tokidney. Peptides were synthesized based on the sequence derived from thebacteriophage which bound selectively to brain. These peptides were ableto block the adherence of the bacteriophage to the brain capillaries.These data suggest that Armored RNA™ can be developed into a deliverysystem for gene therapy or drug treatment which will target specificorgans. Indeed, it has been demonstrated that filamentous bacteriophagedisplaying the peptide sequence RGD, which promotes binding to the cellsurface receptor integrin, are internalized in human HEp-2 cells (Hart,1994).

The Armored RNA™ may also be added to in vitro translation systems suchas a rabbit reticulocyte extract. As stated earlier, capped, polyA mRNAmay used if the reRNA is packaged post-transcription using an in vitropackaging system.

The Coat Protein or the Maturase could be modified to contain thebiotinylation peptide sequence which leads to this peptide becomingbiotinylated in E. coli. In this strategy, the Armored RNA™ would becoated on the surface with biotin molecules which are accessible tostreptavidin. Streptavidin may be conjugated to ligands which recognizespecific cell surface receptors. The ligand-streptavidin conjugate maybe incubated with the Armored RNA™ to coat it with thestreptavidin-ligand conjugate. The ligand-streptavidin-Armored RNA™complex may be used to deliver the packaged RNA.

Antibodies to Armored RNA™ could be conjugated to receptor molecules aspreviously performed (Douglas et al., 1996). The Armored RNA™ particleswould be pre-coated with an antibody-receptor conjugate thus conferringreceptor specificity to the Armored RNA™. The Armored RNA™ AntibodyReceptor complex would bind to a specific cell and then be taken intothe cell.

Armored RNA™ may be made more effective as a delivery vector if theArmored RNA™ disassembles easily once it enters the cell. By usingmutant forms of the Coat Protein in the Armored RNA™, it may be possibleto produce less stable structures which may protect the encapsidatedreRNA from ribonucleases but dissociates readily upon entry into a cell.Mutant forms of the Coat Protein have been studied which are lessefficient at capsid assembly (LeCuyer, 1995) and have less thermalstability compared to the wild-type Coat Protein (Stonehouse, 1993).Higher concentrations of the Coat Protein are needed to induce capsidassembly than for the wild-type sequence. However, Armored RNA™assembled in vivo in the presence of these mutant Coat Proteins may bemore efficient for RNA delivery.

EXAMPLE XIX Production of RNA to Construct an Affinity Column

The need for RNA extends to producing columns for the affinitypurification of proteins which bind to specific RNA sequences. Suchproteins are involved in the regulation of translation and in RNAturnover. Armored RNA™ technology may be used to produce largequantities of a specific RNA sequence. The reRNA would be purified fromthe Armored RNA™ particles. The reRNA could be covalently conjugated toa solid phase resin. Alternatively, a more generic resin may bedeveloped in which Coat Protein was covalently bound to the resin. ThereRNA could be immobilized to the resin by its binding to the CoatProtein through the Operator sequence. The non-phage sequence in thereRNA would then be used to pull out proteins which specificallyrecognize it.

In another format, the protein-reRNA binding could occur in solution, tobetter mimic the actual interaction and then the free reRNA and theprotein-reRNA complexes would be concentrated using a Coat Proteincolumn. The column may be subjected to ribonuclease to release theproteins which bound to the reRNA. These proteins may then becharacterized by gel electrophoresis or the material eluted from thecolumn may be injected into animals such as mice and rabbits to produceantibodies. Once antibodies are produced, then cDNA expression librariesmay be screened for the gene responsible for the protein binding to theRNA.

EXAMPLE XX Packaging of Capped mRNA in vivo

Using Armored RNA™ technology, mRNA may be capped and packaged in E.coli. The enzyme RNA guanyltransferase is responsible for capping the 5′terminus of an mRNA using GTP and S-adenosylmethionine (SAM) assubstrates. RNA guanyltransferase has been cloned and expressed in E.coli in an active form (Cong, 1995). The RNA guanyltransferase may beco-expressed with the RNA to be capped and to be packaged as an ArmoredRNA™ particle. In the preferred embodiment, the non-phage RNA would bepositioned at the 5′ end of the reRNA.

EXAMPLE XXI Encapsulation as a Method For Producing Nuclease-ResistantRNA

“Caging,” or encapsulation of RNA makes an RNA not accessible to anRNase-containing solution. Encapsidation of RNA by viral proteins is anexample of microencapsulation. Microencapsulation is a process whereby amolecule (in this case RNA) is surrounded by a structure that deniesaccess to the internalized molecule. Microencapsulation confersnuclease-resistance upon RNA by sequestering the RNA from thenuclease-contaminated environment. In addition to viral proteinencapsidation, the most common methods of microencapsulation areinternalization in liposomes and inclusion in polymer matrices. Viralprotein encapsidation has proven capable of providing nuclease-resistantRNAs (see Armored RNA above). Liposomes and polymer matrices partitionmolecules in a manner that is analogous to protein encapsidation, thusthey are expected to perform equally well in protecting internalized RNAstandards.

1. Liposomes

The formation of liposomes occurs spontaneously when lipids aredispersed in aqueous solutions. Reagents that are present in the aqueoussolution are encapsulated during liposome formation, providing arelatively simple method for microencapsulating pharmaceuticals, cells,proteins, and nucleic acids. Several methods exist for producingliposomes (Vemuri 1995). The best characterized of these involves addingan excess volume of reagent-containing aqueous buffer to around-bottomed flask in which a lipid solution had been lyophilized. Theaddition of the aqueous solution first dissolves the lipids and thencauses them to form liposomes that engulf portions of the aqueoussolution. This effectively produces liposome-encapsulated reagents thatcan be purified, quantified, stored, and eventually used in diagnosticassays.

2. Polymer Matrices

A variety of monomers can be converted to polymeric microspheres bydropwise dispersion into a solution that encourages polymerization.Molecules dissolved in either the monomer solution or the solution inwhich the polymerization is occurring will be trapped in themicrospheres. Purification of the microspheres provides trapped reagentsthat are inaccessible to macromolecules in the surrounding solution.

An illustration of how to produce reagent-containing microspheres comesfrom a study on the microencapsulation of rotavirus in alginate/sperminematrices (Offit 1994). A rotavirus suspension mixed with sodium alginate(0.68 mM) was dispersed as 5 μm droplets into 0.55 mM sperminehydrochloride. The mixture was centrifuged at 600×g and washed severaltimes to purify the rotavirus containing microspheres. Analysis of themicrospheres revealed that the rotavirus were contained within thepolymer matrix, and that upon release, they were still infectious. Thesetwo points are important as it reveals that microspheres sequestermolecules from the surrounding solution and that the internalizedmolecule is being maintained in its native state. Microencapsulation ofan RNA standard could be done by replacing the rotavirus suspension withan RNA solution.

A single, general method can be employed for incorporatingmicroencapsulated RNAs into a diagnostic assay. RNAs possessing domainssufficient for detection (primer binding or hybridization sites) wouldbe encapsulated by one of the methods mentioned above. The encapsulatedRNA would be purified, quantified, and stored for future use. Fordiagnostics, the microencapsulated standard would be diluted andaliquoted into the samples at an appropriate step. If the standard RNAisolation procedure in the diagnostic protocol does not adequatelyrelease the standard, or if the standard is added after the isolationstep, then heating the sample to 90° C. for 2-5 minutes will besufficient for release from most microcapsules. The RNA detectionportion of the assay could then be performed.

EXAMPLE XXII Noncovalently Bound RNA as a Nuclease-Resistant Standard

RNAs are bound with relatively high affinity by a variety of molecules.In many cases, the RNA can be completely coated by the interactingmolecules. There are examples whereby the presence of the boundmolecules renders the RNA resistant to nuclease degradation. Thisgeneral scheme provides an additional method for generatingnuclease-resistant RNA standards.

There are a variety of proteins, nucleic acids, and small molecules thatbind RNA. Footprinting experiments with HIV-1 nucleocapsid protein(Allen 1996) and the regA protein of T4 (Winter et al 1987) show thatbound molecules can render an RNA resistant to nucleases. Proteins andsmall molecules that bind RNA cooperatively often coat large regions ofthe polymers. Examples include the T4 gene 32 protein (von Hippel 1982),MS2/R17 coat protein (Witherall 1990), and the small molecules spermineand spermidine (McMahon 1982). The combination of cooperative bindingand nuclease occlusion can effectively protect large domains of RNAsfrom degradation (Allen 1996) making them ideal nuclease-resistantstandards. Poly-L-lysine is able to protect the natural double-strandedRNAs interferon inducers, larifan and ridostin, from ribonucleases inhuman blood serum. The free interferon inducers in serum completely losttheir activity after 4 hour incubation in serum. The protective activityof poly-L-lysine increased in parallel with the increase in itsmolecular weight. The protective activity was maximal with material of12,300±1,000 Daltons (Surzhik et al., 1993).

Cetyltrimethylammonium bromide (CTAB) is a cationic detergent which hasbeen long used for the isolation of nucleic acids by virtue of itsability to bind to and precipitate nucleic aids but not protein (Jones,1953; Jones, 1963). The positively charged head groups can bind to thenegatively charged backbone of the RNA, protecting the RNA fromribonuclease. Experiments by Winkler and Kessler (unpublished)demonstrate that 20 mM CTAB protects as much as 5 micrograms RNA fromdigestion by RNase A and RNase T1. If 0.5% Triton X-100 is added to theCTAB/RNA solution, the CTAB is solubilized by the Triton X-100 and theprotective action is reversed.

Similarly, nucleic acids afford nuclease protection by directlyinteracting with RNA (Weigand 1975). Short (ten nucleotides), randomsequence nucleic acids can be hybridized to an RNA, effectively coatingthe molecule. Incubation in a sample that includes RNases would not bedetrimental, as the RNA would be bound by the randomers, and thusprotected. If an amplification scheme is used for detection, the shortrandomers could serve as primers for reverse transcriptase.

A general scheme can be devised by one of skill for employing any of thenoncovalently bound RNAs as standards. An RNA with sequences for primerbinding (for RT-PCR, NASBA, or 3SR) or probe hybridization (branched DNAassay or DiGene's antibody based assay) can be prebound by any of theRNA binding molecules mentioned in the preceding paragraphs. This boundRNA would be accurately quantified and stored for future use. The boundRNA would be diluted, and aliquoted into samples, either before or afterRNA purification. Prior to amplification or hybridization (depending onthe detection protocol), the sample would be heated to 90° C. to releasethe RNA standard. The amplification or hybridization could then beperformed, producing a signal for detection.

EXAMPLE XXIII RNA Bound by gp32 is Nuclease-Resistant

An RNA bound by the protein encoded by gene 32 of the bacteriophage T4(gp32) was used to demonstrate the effectiveness of noncovalent bindingmethodology for making RNA standards. gp32 is known to bind RNA andssDNA cooperatively (von Hippel 1982). The purified protein (˜1 μM) wasmixed with a radiolabeled RNA target (˜100 nM) for five minutes at roomtemperature in a buffer consisting of 50 mM HEPES pH 7.4, 200 mM NaCl,and 2.5 mM MgCl₂. By gel shift assay, approximately 50% of the RNAmolecules were bound by the protein. A tenth volume of goat serum wasadded to the coated RNA and the mixture was incubated at 25° C. for tenminutes. The RNA was separated via PAGE and detected by autoradiography.The amount of intact RNA was greater than an identical RNA sample thatlacked the protein. This illustrates that the presence of gp32 protectsRNA from serum-mediated degradation and argues that the concept ofnuclease protection generated by cooperatively bound molecules is valid.

A sample of the same RNA used above was bound by gp32 using thedescribed procedure. This sample was heated to 95° C. for 2 minutes andthen reverse transcribed by AMV reverse transcriptase using a primerspecific to the 3′ end of the RNA. The resulting cDNA was seriallydiluted and the various dilutions were used to initiate a standard PCRreaction using primers specific to the RNA standard. The PCR productswere analyzed by gel electrophoresis. The dilution at which product wasfirst observed was the same for the gp32-bound RNA as for an unbound RNAsubjected to the same procedure. This indicates that the T4 protein doesnot affect the capacity of RT to use the RNA as a template, nor does itsubsequently affect the PCR reaction. Protein-coated RNAs have also beendiluted into a 10% serum solution for fifteen minutes and then appliedto an RT-PCR experiment. Products of the appropriate size weregenerated, providing evidence that cooperative binding of an RNA is auseful method for producing stable RT-PCR controls.

EXAMPLE XXIV Protection of RNA through Chemical Modification

One method for making RNAs resistant to enzyme-mediated degradation isto chemically alter the RNA so that the mechanism for degradation isblocked or so that RNases no longer recognize the molecule as RNA. Suchalterations can be made to nucleotides prior to their incorporation intoRNA or to RNA after it has been formed. Ribose (Piecken 1991) andphosphate (Black 1972) modifications have been shown to enhance RNAstability in the presence of nucleases. Modifications of the 2′ hydroxyland internucleotide phosphate confers nuclease resistance by alteringchemical groups that are necessary for the degradation mechanismemployed by ribonucleases (Heidenreich 1993). Another method could beaccomplished by chemically adding molecules (e.g., carbohydrates) to theRNA standard. These chemical groups could be removed enzymatically orchemically during the detection protocol to allow for reversetranscription. Provided that they are templates for the reversetranscription or hybridization protocols used in nucleic acid detectionschemes, chemically modified RNAs will be ideally suited as RNAstandards as they are considerably more stable than non-modified RNA.

The general scheme for using chemically modified RNAs as standards indiagnostic assays is to simply replace the current RNA standards withmodified RNAs possessing the same sequence. Transcribe the DNA templateencoding the RNA standard using T7 RNA polymerase, replacing thestandard NTPs with ribose or phosphate modified NTPs. The resultingfull-length RNA can be purified, quantified and stored for future use.At the time of the assay, the modified RNA standard can be appropriatelydiluted, aliquoted into sample either before or after RNA purification,and then co-detected with the sample RNA.

EXAMPLE XXV Protection of RNA With 2′ Fluoro CTP and UTP

To test the usefulness of modified RNAs as standards, nucleotide analogswere incorporated into transcripts and compared to unmodified RNAs. 2′fluoro CTP and UTP were incorporated into transcripts by 0.5 units/μl ofT7 RNA polymerase in transcription buffer (40 mM Tris pH 8.0, 20 mMNaCl, 6 mM MgCl₂, 6 mM MnCl₂, 2 mM spermidine, 10 mM DTT). Ten picomolesof modified and unmodified RNA were reverse transcribed by AMV reversetranscriptase. A dilution series of the resulting cDNAs were amplifiedby PCR. The products were separated via PAGE and analyzed by ethidiumbromide staining. Both the modified and unmodified RNA samples generatedidentical results, indicating that the modifications have no effect onthe quantification of the RNAs by RT-PCR. The modified RNAs wereincubated in serum for one hour and the products were size-separated byPAGE. No degradation of the modified RNAs was apparent, while anunmodified RNA of the same sequence was completely degraded. Thus, themodified RNAs would be far superior to unmodified RNA as they could beadded earlier in the diagnostic protocol (providing a better internalstandard) and they would be more stable during storage.

The ultimate test of the modified RNA is to show its compatibility withan existing diagnostic assay. A template encoding the sequence of thestandard for the Amplicor HIV Monitor™ assay was transcribed with 2′FCTP and UTP as above. A series of dilutions of the 2′ F modified RNA wasapplied to the Amplicor HIV Monitor™ test. The modified standardproduced a signal in a concentration dependent fashion, providingevidence that the modified RNA was being reverse transcribed and thatthe resulting cDNA was being PCR amplified. The modified RNA standardwas then compared to an unmodified RNA. Both standards producedapproximately equal signals when introduced after the lysis step of thedetection protocol, but when the two RNAs were incubated in plasma forfifteen minutes prior to incorporation into the detection protocol, thesignal produced by the unmodified RNA was indistinguishable frombackground whereas the modified RNA produced signals that were nearlyequivalent to is that observed when the RNA was not preincubated inplasma. This latter observation points to the obvious advantage of themodified RNA standard, namely that the RNA can be added earlier in theprotocol (prior to sample lysis) providing a better control for theoverall experiment. Additionally, nuclease-resistance will be beneficialduring storage, as the likelihood of degradation prior to assaying willbe greatly diminished.

Phosphate modified nucleotides, in the form of a-thiols of CTP and UTP,were incorporated into transcripts by T7 RNA polymerase as the 2′ Fabove. The resulting RNAs were templates for reverse transcriptase. PCRanalysis revealed that the modified RNAs were converted to cDNAs asefficiently as unmodified RNAs.

Although the work described used only modified pyrimidines, modifiedpurines can also be incorporated into RNA [Aurup et al. (1994)]. Thismight be important in a case where a high concentration of apurine-specific nuclease is present in a group of samples. Because avariety of nucleotide analogs exist that can be incorporated, RNAs canbe tailored for different storage and reaction conditions to ensureoptimum stability and assay efficiency.

EXAMPLE XXVI Assay for Ribonuclease Protection

To test a potential ribonuclease-resistant construct for ribonucleaseresistance, a protected RNA construct is incubated with ribonuclease,such as Ribonuclease 1 or Ribonuclease A, at 37° C. for severaldifferent time points, 15 minutes, 30 minutes, 1 hour, 2 hours. Duringthe same experiment, naked RNA isolated from the same Armored RNAconstruct is also incubated with the ribonuclease. After each timeperiod, the ribonuclease is inactivated by phenol extraction. The phenolalso strips the coat protein from the RNA in an Armored RNA construct.After phenol extraction, the RNA is ethanol precipitated. The isolatedRNA is fractionated on a denaturing formaldehyde agarose gel, stainedwith ethidium bromide and assessed for degradation. Degradation isassessed by comparing the ribonuclease treated samples to RNA which wasnot ribonuclease treated.

Alternatively, instead of analyzing the RNA by gel electrophoresis, asample can be subjected to competitive RT-PCR to obtain a more accurate,quantitative result. The use of PCR also has the advantage of detectingsmall amounts of material.

EXAMPLE XXVII Standard Steps in Using Any RNase Resistant Standard

Many assays employing RNase resistant standards will have the same basicformat as the Amplicor HIV Monitor™ assay described above. In such akit, known amounts of RNase resistant standard is added to the RNAsample. The sample RNA and the RNA standard (eg., Armored RNA™,chemically modified RNA or any other ribonuclease protected RNA) areco-purified from plasma or from cells. For some assays, the sample RNAwill already have been purified. In such cases, the RNase resistantstandard may be added directly to the sample RNA. The mixture can thenbe heated for 5 minutes, 70° C. to strip off a protein coat, or anyother molecule that confers ribonuclease resistance, or treated in anyother manner necessary.

After the RNA has been purified or heated, the sample RNA and thestandard RNA are reverse transcribed and then co-amplified by PCR. Ifthe standard RNA has a deletion or insertion, then the PCR products maybe fractionated on a gel. The PCR product generated from the RNAstandard will have a different mobility than the PCR product derivedfrom the sample RNA. A standard curve can be generated plotting theconcentration of RNA standard and the amount of PCR product generatedfrom the standard. The concentration of the sample RNA is derived byinterpolation within the linear range of the curve.

In the HIV Monitor™ assay, the RNA standard has a 26 nucleotidesubstitution compared to the sample RNA sequence. Quantification of thePCR products are derived by using immobilized oligonucleotides tospecifically capture the PCR products by hybridizing to the test PCRproduct or the standard PCR product. The PCR products are 5′biotinylated. The captured PCR products are detected colorimetricallyusing streptavidin horseradish peroxidase. Once again, the concentrationof the unknown RNA sample is calculated using the signal produced by theknown concentration of the standard RNA.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the compositions, methodsand in the steps or in the sequence of steps of the methods describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physically related may be substituted forthe agents described herein while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-71. (canceled)
 72. A method of detecting and/or quantifying a cellularmRNA in a sample comprising: (a) obtaining a sample; (b) obtaining aribonuclease resistant RNA standard comprising a recombinant RNA segmentstandard encapsidated in a viral coat protein, wherein the recombinantRNA segment standard comprises a region encoding a sequence that can bea standard for detecting and/or quantifying the cellular mRNA; (c)employing the recombinant RNA segment standard as a standard to detectand/or quantify the cellular mRNA in the sample; and (d) detectingand/or quantifying the cellular mRNA in the sample.
 73. The method ofclaim 72, wherein the sample is a tissue sample.
 74. The method of claim72, wherein the sample is a blood sample.
 75. The method of claim 72,wherein the cellular mRNA encodes a cytokine, a cell cycle gene, or anoncogene.
 76. The method of claim 75, wherein the sequence that can be astandard for detecting and/or quantifying the cellular mRNA is furtherdefined as a sequence that can be a standard for detecting and/orquantifying a cytokine.
 77. The method of claim 76, wherein the sequencethat can be a standard for detecting and/or quantifying the cytokinecomprises a modified cytokine sequence.
 78. The method of claim 75,wherein the sequence that can be a standard for detecting and/orquantifying the cellular mRNA is further defined as a sequence that canbe a standard for detecting and/or quantifying a cell cycle gene. 79.The method of claim 78, wherein the sequence that can be a standard fordetecting and/or quantifying the cell cycle gene comprises a modifiedcell cycle gene sequence.
 80. The method of claim 75, wherein thesequence that can be a standard for detecting and/or quantifying thecellular mRNA is further defined as a sequence that can be a standardfor detecting and/or quantifying an oncogene.
 81. The method of claim80, wherein the sequence that can be a standard for detecting and/orquantifying the oncogene comprises a modified oncogene sequence.
 82. Themethod of claim 72, further comprising isolating the RNA segmentstandard from the viral coat protein.
 83. The method of claim 72,further comprising isolating sample RNA from the sample.
 84. The methodof claim 83, further comprising isolating the RNA segment standard fromthe viral coat protein.
 85. The method of claim 84, further comprisingadmixing the sample and the standard prior to isolating the RNA segmentstandard so that isolation of the sample RNA and isolation of the RNAsegment standard from the viral coat protein are performed in the sameisolation procedure.
 86. The method of claim 72, further comprisingadmixing the sample and the standard prior to quantifying the cellularmRNA.
 87. The method of claim 72, wherein the standard used to detectand/or quantify the cellular mRNA in the sample is an internal standard.88. The method of claim 72, wherein the standard used to detect and/orquantify the cellular mRNA in the sample is an external standard. 89.The method of claim 72, wherein the standard used to detect and/orquantify the cellular mRNA in the sample is a positive control standard.90. The method of claim 72, wherein the method is a detection assay. 91.The method of claim 72, wherein the method is a quantification assay.92. The method of claim 72, wherein the method is a detection andquantification assay.
 93. The method of claim 72, wherein the methodfurther comprises using a polymerase chain reaction procedure.
 94. Themethod of claim 93, wherein the polymerase chain reaction procedurecomprises: employing reverse a reverse transcription procedure;amplifying a DNA to generate a polymerase chain reaction product; anddetecting the polymerase chain reaction product.
 95. The method of claim94, wherein amplifying comprises co-amplification of a polymerase chainreaction product amplified from the sample RNA and a polymerase chainreaction product amplified from the RNA segment standard.
 96. The methodof claim 95, wherein the co-amplification involves the use of a singleprimer set for amplification of both the polymerase chain reactionproduct amplified from the sample RNA and the polymerase chain reactionproduct amplified from the RNA segment standard in a reversetranscriptase-polymerase chain reaction procedure.
 97. The method ofclaim 95, wherein the method comprises comparing an amount of polymerasechain reaction product amplified from the sample RNA with an amount ofpolymerase chain reaction product amplified from the RNA segmentstandard.
 98. The method of claim 72, wherein the viral coat protein isa modified coat protein having a different amino acid sequence ascompared to a native sequence.
 99. The method of claim 72, wherein theviral coat protein comprises bacteriophage viral coat protein.
 100. Themethod of claim 99, wherein the bacteriophage viral coat protein is ofan MS2/R17 bacteriophage.